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juniperpublishers-nutrition · 2 years ago

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Antiproliferative Effect of Green Papaya on Lymphocytic Leukemic Cells

Abstract

Numerous edible plants have been reported to interfere with the carcinogenic process, and therefore, the regular consumption of these plant products may reduce the risk of developing cancer. We investigated the effect of papaya fruit and leaves on the cell proliferation of Jurkat T-lymphocytic and Daudi B-lymphocytic leukemia cells. Cells were treated with aqueous or methanolic extracts from leaves, skin, pulp, and seeds from green papaya. The papaya fractions were tested for total phenolic content, total flavonoids content, and anti-oxidation activity using chemical assays. Cell proliferation was measured using a WST-1 assay. Our data indicate that methanol and water extracts of seeds and leaves contained higher concentrations of total phenolic compounds and higher anti-oxidation activity than that of extracts from skin and pulp.

Both methanol and water extracts from leaves and skin potently inhibited the proliferation of leukemic Jurkat T-cells and Daudi B-cells. However, the effect was more potent on Jurkat T-cells, and the leaf extracts were more effective than that of skin extracts. None of the pulp or seed extracts showed inhibitory activity on leukemic cell proliferation. Although papaya leaves are not consumed as a food, leaf extracts have been used for the treatment of various conditions, including dengue and malaria fevers, gastric ulcers, low platelet counts, and cancers of the breast, lung, and cervix. Our data suggest that the consumption of papaya leaf extracts may also be beneficial in preventing and/or treating lymphocytic cancer. Isolation of active compounds from papaya leaves will also help in developing new drugs for cancer treatment.

Keywords: Polyphenols; Antioxidants; Anticancer; Jurkat cells; Daudi cells; Leukemia

Introduction

Leukemia is a cancer of the blood or bone marrow, as described by an anomalous multiplication of immature clonal hematopoietic cells. Leukemia is one of the cancers that can affect all races in the United States. The American Cancer Society estimates that there will be around 60,530 new cases of leukemia in the United States in 2020, resulting in 23,100 deaths [1]. Leukemia is classified based on its onset (acute or chronic), the affected blood cell type (lymphoblastic/lymphocytic or myeloid/myelogenous), the maturity stage of the blood cell, and phenotypic expression of the disease [2]. Leukemia, like other forms of cancer, can be treated by using a number of methods, including chemotherapy, radiation therapy, targeted therapy, or stem cell therapy, though it is easier to treat acute leukemia than chronic leukemia [3]. Although leukemia remains a common medical issue, there has been a decrease in incidence due to preventive measures, and death rates have also declined since 1991 due to recent advances in treatment strategies [4]. The 5-year survival rate for children with acute lymphocytic leukemia has greatly increased over time to about 90% [5]. Although there are improved treatment strategies and survival rates, the cost of such therapies remains an issue that hinders access for all patients [6]. There is a need for less expensive alternative strategies to increase treatment access and survival outcomes for all patients.

Recent advances in alternative and complementary medicine have shown that various herbs, fruits, and vegetables have compounds that could act as inhibitors of cancer formation, blockers of carcinogen interaction, and suppressors of tumor progression. For example, Korean red ginseng extract treatment inhibited the growth of U937 leukemia cells by downregulating the expression of human telomerase reverse transcriptase [7]. Another study confirmed that Kenaf seed oil causes the death of human leukemia cells HL60 and K562 by induction of apoptosis [8]. Studies demonstrated that a hot water extract of Euphorbia formosana (EFW) restrained the growth of THP-1 human leukemic cells by inducing apoptosis through caspase-dependent cell death via Fas and mitochondrial pathways [9]. Water, ethanol, and methanol extracts of garlic have exhibited lethal cytotoxic effects of 50-75% on cells harvested from 3 patients with acute lymphoblastic leukemia (ALL) and three patients with acute myeloid leukemia (AML) [10]. Typhonium flagelliforme (TF), a herbal plant in Malaysia, exhibited cytotoxic effects in P388 [11] and WEHI-3 [12] murine leukemia cells. Further, in vivo studies showed a significant decline in the cell count of immature monocytes and immature granulocytes, when the TF extract was orally administered for 28 days in a BALB/c leukemic mouse model [12]. Studies with grape seed extracts reported induction of apoptosis in Jurkat leukemic cells through a process that involves sustained JNK activation and Cip1/p21 up-regulation, culminating in caspase activation [13].

Papaya fruit has also been studied for its anticancer activities for colorectal [14], prostate [15,16], cervical [17], breast [18- 20], and skin cancers [21]. Papaya also effectively inhibits cancers of hematopoietic cell lines, including T cell lymphoma (Jurkat), plasma cell leukemia (ARH77), Burkitt’s lymphoma (Raji), anaplastic large cell lymphoma (Karpas-299), and human promyelocytic leukemia (HL-60) [22-23]. A study demonstrated that Benzyl isothiocyanate (BITC), a promising chemo preventive agent found in papaya fruits, when given followed by cisplatin (BITC/cisplatin) caused significant death in HL-60 cells [24]. Furthermore, a recent study reported that papaya leaf extract improved bone marrow blast counts and stabilized the hematological parameters of a 76-year-old male with untreated chronic myelomonocytic leukemia [25].

It is well Known that fruits, vegetables, and other medicinal plants contain polyphenols and flavonoids that exhibit various biological and pharmacological properties [26], which are often mediated by their anti-oxidation activity [27]. Based on these earlier studies, we evaluated water and methanol extracts from leaves, skin, pulp, and seeds of green papaya on T-lymphocytic leukemia and B-cell lymphocytic leukemia using Jurakt and Daudi cell lines, respectively, and hypothesized that the effect of papaya fractions on cell proliferation of leukemic cells is correlated to their phenolic contents and/or anti-oxidation activity.

Materials and Methods

Materials

The Jurkat T-cell line and Daudi B-cell line were purchased from ATCC (Manassas, VA). RPMI 1640 medium was purchased from Gibco (Grand Island, NY). Fetal Bovine Serum (FBS) was purchased from RAMBIO (Missoula, MT). Penicillin and streptomycin antibiotics (BP2959) and Phosphate Buffered Saline (PBS; BP399-550) were purchased from Fisher Scientific (Fair Ln, NJ). Folin Ciocalteu, sodium carbonate, aluminum chloride, Diphenyl-1-picrylhydrazyl (DPPH), quercetin, gallic acid, and trolox were purchased from Sigma Chemical Co. (St Louis, MO). WST-1 (MK400) was purchased from Takara (Kusatsu, Shiga, Japan). Green papaya was obtained from Randolph Farm at Virginia State University, Petersburg, VA.

Isolation of papaya leaves, skin, pulp, and seeds

The papaya samples were isolated as described previously [28]. Briefly, the papaya was washed with distilled water and blotted dry with a paper towel. The skin was peeled off using a kitchen peeler. An unskinned papaya was cut into half to remove seeds, and then the pulp was cut into small pieces. The leaves and seeds were washed with distilled water. All fractions (leaves, skin, pulp, and seeds) were freeze-dried. The dried leaves, skin, pulp, and seeds were ground to a fine powder using a mortar and pestle with liquid nitrogen added to keep the powder frozen. The dried powder was flash-frozen with liquid nitrogen and stored at -80 ℃ until used.

Preparation of extracts from papaya leaves, skin, pulp and seeds

The methanol and water extracts of papaya fractions were prepared as described previously [28]. Briefly, a known quantity (5g) of different fractions of freeze-dried papaya powder was homogenized with 200ml of 80% methanol or distilled water. The mixtures were placed on a shaker at room temperature overnight. The next day, the mixture was centrifuged at 2000 x g for 20 minutes using a Thermo Scientific centrifuge (Waltham, MA). The supernatants were collected, and the residues were washed two times by resuspending in the respective solutions, mixing, and placing on a shaker overnight. The collected supernatants were pooled, and the insoluble residues were discarded. The methanol extracts were dried in a nitrogen evaporator (Organomation Associates, Inc, Berlin, MA), whereas the water extract was freezedried. The dried extracts were stored in a -20℃ freezer. The dried extract was suspended into DMSO at a concentration of 250mg/ ml and was further diluted into media for cellular studies.

Characterization of anti-oxidation activity of papaya extracts

The oxidative properties of papaya were determined by assaying their total polyphenolic content (TPC), total flavonoid content (TFC), and anti-oxidation capacity (DPPH) as previously described [29].

Cell culture

Jurkat T-cells and Daudi B-cells were maintained in RPMI 1640 media supplemented with penicillin (100 units/ml), streptomycin (100μg/ml) and 10% FBS. All cell cultures were incubated in a humidified incubator at 37 °C and 5% CO2. Media was changed every three days, and cells were subcultured when they became confluent.

Cell proliferation assay

The effects of papaya leaf, skin, pulp, and seed extracts on cell proliferation were determined using a WST-1 assay as per manufacturer instructions and described previously [28]. Briefly, cells were isolated from a confluent flask, and about 10,000 cells were placed in each well of a 96 well plate and incubated in a CO2 incubator for 24 hours. The next day, media was removed, and cells were incubated in serum-free media containing varying concentrations of papaya leaf, skin, pulp, or seed extract for 24 hours. After treatment with papaya extract, 10 μl of WST-1 was added in each well, and the plate was read at 420 nm in a 96 well plate reader for the background reading. The plate was then incubated for 3 hours at 37 ℃ in a CO2 incubator, and the reduction of WST-1 dye due to mitochondrial oxidation was recorded at 420 nm. The change in absorbance was calculated by subtracting the initial reading from the final reading. The results are expressed as % change from control (untreated cells).

Data analysis

The data are expressed as mean ± SD for at least three replicates. All comparisons were made by one-way ANOVA with Tukey’s -HSD-post-hoc test using SPSS Statistics 20 software (IBM, Armonk, NY). All significant differences are reported as P < 0.05 and indicated by “*” whereas non-significant differences are not marked.

Results

Characterization of papaya extract for anti-oxidation activity

The anti-oxidation potentials of papaya extracts were determined by assessing their total polyphenol content, total flavonoid content, and by assaying their anti-oxidation capacity. The data showing the total phenolic contents are presented in Table 1 and reported previously [28]. The highest amounts of polyphenols were found in the seed extracts that ranged 14 -16 mg/g dry weight. There was no significant difference in total phenolic contents between water and methanol extracts from papaya seeds. The leaves were second highest in total phenolic content but had a significantly lower amount of total phenolic content than that of seeds (P < 0.05). The leaves contained 1- 4 mg/g dry weight total phenolic content. Water extracts of leaves contained less total phenolic content (~ 1 mg/g dry weight) compared to that of methanol extracts. The skin extracts contained total phenolic content ~ 0.5 - 1 mg/g dry weight, whereas the pulp extracts have very small amounts of total phenolic content (0.05 - 0.5 mg/g dry weight).

The data for the total flavonoid contents in various papaya fractions are presented in (Table 2) and reported previously [28]. The water extract of seeds contained the highest amounts of total flavonoid content (5 mg/g dry weight), whereas the methanol extracts contained lower amounts (2-2.5 mg/g dry weight, P < 0.05). In leaves, the methanol extract contained more total flavonoid content (~ 5mg/g dry weight) than that of water (~2 mg/g dry weight). The total flavonoid contents in pulp and skin were less than those of seeds and leaves. In the skin, higher total flavonoid contents were present in the methanol extracts (~ 3.2 - 3.5 mg/g dry weight) than in water extracts (1 mg/g dry weight). The pulp contained the lowest total flavonoid contents compared to other fractions. The total flavonoid content in pulp ranged from 1 - 1.5 mg/g dry weight.

The anti-oxidation capacities of papaya fractions were measured by assaying the inhibition of DPPH oxidation and are shown in (Table 3) and reported previously [28]. The seeds and leaves contained significantly more (P < 0.05) anti-oxidation capacity than that of the skin and pulp. The methanol fractions of seeds and leaves contained more anti-oxidation activity than water extracts. The methanol fractions of seeds and leaves inhibited DPPH oxidation by 75 - 85%, whereas the water extracts of these fractions inhibited DPPH oxidation by 50 - 70%. The skin and pulp inhibited DPPH oxidation from 25 - 35%. There were no significant differences between water or methanol extracts of skin and pulp.

Effect of papaya water extracts on T- cell leukemia

The effects of papaya water extracts from different fractions are presented in Figure 1. The treatment with leaf extract caused a dose-dependent decrease in cell viability. A concentration of the extract as low as 50 μg/ml caused significant cell death by 30% (P < 0.05). Upon increasing the extract concentration more cells died, reaching a plateau of almost 70% cell death (P < 0.05) at 150 μg/ml. Additional increases to 250 μg/ml concentration resulted in a very small change in cell viability (Figure 1).

The treatment with water extract from papaya seeds caused a dose-dependent decrease in cell viability. Extract concentrations as low as 150 μg/ml caused a significant 20% increase in cell death (P < 0.05). On further increasing the extract concentration, no further decrease in cell viability occurred, which reached a plateau at 250 μg/ml with 25% cell death (P < 0.05) (Figure 1)

The water extract of papaya skin started to significantly (P < 0.05) inhibit leukemic cell proliferation at 25 μg/ml, causing about 30% cell death. However, the leukemic cells progressively died in a dose-dependent manner reaching to about 80% cell death at 250 μg/ml of the dried papaya skin extract.

The water extract from papaya pulp was unable to cause significant cell death of Jurkat leukemic T-cells.

Effect of papaya methanol extracts on T- cell leukemia

The effects of papaya methanol extracts from different fractions are presented in Figure 2. The methanolic extract of papaya leaves appeared to be less effective than water extract. At 25 μg/ml a significant (P < 0.05) 30% decrease in cell viability resulted; however, the cell viability was decreased by only 60% (P < 0.05) on further increasing the methanolic extract concentration to 250 μg/ml. Similarly, the methanolic extract of papaya seeds appeared to be less effective than water extract. At 200 μg/ml, a significant P < 0.05) decrease in cell viability resulted (10%); however, the further decrease in cell viability was not significant at 250 μg/ml. The methanolic extract of skin also caused significant cell death by 25% at 25 μg/ml (P < 0.05). Further increase of the extract concentration resulted in a dose-dependent response reaching 80% cell death at 250 μg/ml .

The methanol extract from papaya pulp caused a modest significant effect by decreasing cell viability by 20% at 50 μg/ml ; however, on increasing the concentration of pulp extract to 250 μg/ml , there was no further increase in cell death.

Effect of papaya water extracts on B- cell leukemia

The effects of water extract from different papaya fractions on Daudi leukemia B-cells are presented in Figure 3. Treatment with leaf extract caused a dose-dependent decrease in cell viability. Concentrations as low as 25 μg/ml caused a significant 20% cell death (P < 0.05). On further increasing the extract concentration, significantly more cells (P < 0.05) died in a dosedependent manner, with approximately 50% cells dead at the highest concentration of 250 μg/ml. The water extracts from seeds and pulp have no significant effect on cell viability at any concentration. The extract of papaya skin started to significantly (P < 0.05) inhibit leukemic cell proliferation at 25 μg/ml causing about 25% cell death. However, on further increasing the extract concentration, no change in cell death was observed

Effect of papaya methanol extracts on B- cell leukemia

The effects of methanol extracts from different papaya fractions on Daudi leukemia B-cells are presented in Figure 4. The methanolic extract of papaya leaves appeared to affect Daudi B-cells, similar to water extracts. At 50 μg/ml, a significant 30% (P < 0.05) of cells died and the cell death was further increased in a dose-dependent manner to 80% (P < 0.05) at 250 μg/ml. Consistent with the water extract, the methanol extract of papaya pulp has no effect on Daudi B-cells. The methanolic extract of skin had a similar effect on Daudi B-cells as the water extract. It caused significant cell death by 25% at 25 μg/ml (P < 0.05). Increasing the concentration of extract resulted in a dose-dependent effect, reaching 50% cell death at 250 μg/ml.

Discussion

The present study was carried out to investigate the effect of water and methanol extracts of papaya leaves, skin, pulp, and seeds on Jurkat leukemia T- cells and Daudi leukemia B-cells. Water and methanol were used because phenolic compounds have different chemical characteristics and polarities, and because of these properties, compounds in different fractions of papaya may or may not be soluble [30]. We initially tested these fractions for their phenolic contents and their anti-oxidation activity. Most of the anti-oxidation activity in fruits and vegetables is due to their total polyphenolic contents [31]. Our data indicate that papaya seeds have higher amounts of polyphenols than that of other fractions (Table 1). Papaya leaves also contained substantial amounts of total phenolic contents but were 1/3 that of seeds. Other fractions have very little total phenolic content. In seeds, the amount of total phenolic content was similar in both extracts, but the leaf water extract has less total phenolic content than that of methanol. However, the water extract of skin contained almost twice the polyphenols than that of the methanol extract. Organic solvents are generally used to extract bioactive agents from fruits and vegetables. The most suitable solvents for the extraction of polyphenols are aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate. Ethanol has been known as a good solvent for polyphenol extraction and is safe for human consumption. Methanol has been generally found to be more efficient in the extraction of lower molecular weight polyphenols, whereas aqueous acetone is good for the extraction of higher molecular weight flavonoids [32]. Currently, over 8000 phenolic structures have been identified in fruits and vegetables [33], and flavonoids are one of the major phenolic classes comprising almost 4000 compounds present in different edible plants [33]. We also determined the total flavonoids in the papaya extracts. Our data show that water extracts of seeds and methanol extracts of leaves contained the highest amount of total flavonoid content, which represented about 30- 40% of total phenolic compounds in seeds and leaves (Table 2). The data suggest that most of the polyphenols in seeds are water-soluble flavonoids, whereas a higher proportion of polyphenols in leaves are less water-soluble flavonoids.

Next, the anti-oxidation activities were determined in these fractions. The leaves and seeds possess a higher anti-oxidation activity compared to that of other fractions (Table 3). Although leaves and seeds have different phenolic and flavonoid contents, they have similar profiles for their anti-oxidation activity. It was interesting to note that methanol extracts exhibited higher antioxidation activity than the water extracts. This difference could be due to the different natures of polyphenols.

Next, we analyzed the anticancer properties of papaya fractions on Jurkat cells and Daudi cells. The Jurkat cells are an immortalized line of human T lymphocyte cells that are often used to study acute T cell leukemia. The Jurkat cell line was established in the late 1970s from the peripheral blood of a 14-year-old boy with T cell leukemia [34] whereas the Daudi cell line is a wellcharacterized B lymphoblast cell line which was derived from a 16-year-old black male with Burkitt’s lymphoma [35].

A large number of phytochemicals from vegetables, fruits, and grains have been reported to possess anticancer properties and are promoted for cancer prevention and treatment [36-40]. Recently, multiple scientific reports have emerged about the medicinal benefits of papaya [16,41-43].

Our data indicated that none of the extracts from seeds or pulp possess significant inhibitory activity for cell proliferation in Jurkat leukemic T-cells or Daudi leukemic B-cells. The extracts from leaves and skin, however, showed significant inhibition of both Jurkat leukemic T-cells and Daudi B-cell growth. This effect was stronger on Jurkat T-cells and modest on Daudi B-cells. During the present study, we found that papaya seeds contained the highest amounts of phenolic compounds; however, none of the extracts from papaya seeds have any inhibitory activity for Jurkat leukemic cells. Our data clearly indicated that the inhibitory activity of these extracts was neither dependent on their phenolic contents, nor was it dependent on their anti-oxidation activity. It appears from this observation that the inhibitory compounds in papaya leaves and skin extracts acted on cellular mechanisms linked to cell growth. For example, phytochemicals have been shown to interfere with the stabilization of the microtubule structure, thereby inhibiting mitosis and cancer cell propagation [44]. Vincristine and vinblastine, isolated from the leaves of Madagascar periwinkle, were the first phytochemicals to be used clinically in combination with other anticancer agents in lymphomas, leukemias, and breast, lung, and ovarian cancers [45,46].

Epigallocatechin-3-gallate (EGCG), a polyphenol from the leaves of Camellia sinensis (processed to green tea), possesses anti-oxidation activity but it limited cancer cell proliferation by reducing DNA methylation through the inhibition of DNA methyltransferase together with reactivation of the silenced tumor suppressor genes [47]. Curcumin (diferuloylmethane), a polyphenol isolated from the rhizome of the turmeric plant, has also shown therapeutic efficacy on numerous disorders, including cancer [48]. Curcumin is reported to inhibit NF-κB signaling that triggers the intracellular inflammatory response as well as cellcycle- associated genes. By arresting the cell cycle and inducing apoptosis through the relaying pathways, curcumin interferes with angiogenesis and reduces tumor invasion.

An additional group of anticancer phytochemicals functions as inhibitors of topoisomerase I or II, which are the nuclear enzymes that control DNA supercoiling, eliminate tangles in the chromatin structure, and allow DNA to be replicated and transcribed. Thus, topoisomerase inhibitors can act as anticancer agents by inducing a delay of the cell cycle, followed by cell death [36]. β-Lapachone from the bark of the Lapacho (Handroanthus impetiginosus) plant, camptothecin from the bark/stem of Camptotheca acuminata (the Chinese happy tree), and podophyllotoxin from the root of the Mayapple (Podophyllum peltatum) plant are examples of phytochemicals inhibiting topoisomerases in cancer cells [49- 51]. We have not yet characterized the chemical composition of aqueous and methanolic extracts of leaves and skin or explored the molecular mechanisms that may be involved in inhibition of Jurkat leukemic cell growth. Clearly, additional experiments are required for isolation and identification of active compounds in papaya leaves and skin to determine the cellular mechanisms of their anticancer activity.

Our data suggest that consumption of papaya leaves or skin may be beneficial for preventing and/or treating leukemia, particularly T-cell leukemia. Historically, papaya leaves have been used in different cultures around the world, which suggests that the consumption of leaves extract is not toxic [52]. It is possible that anticancer compounds from leaves can be extracted by boiling them in water. However, the skin is not typically consumed because of its bitter taste. The skin can be dried and made into a powder that can be consumed in the form of pills. Alternatively, the active compounds from leaves and skin can be extracted and isolated in pure form, which can be the basis for new therapeutic leukemia drugs. In conclusion, this study suggests that leaves and skin of papaya contained anticancer activity for leukemic cells.

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justlemmeadoreyou · 8 months ago

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3. protectively watchful (restaurant owner!harry x chef!reader)

(part 1 here) | (part 2 here)

summary: you take up on the mantorship offer, but it creates more tensions and turmoil within you than were before. an incident in the kitchen makes harry go into protective mode, and you can't help but get turned on by this man more and more.

words: 4.8k

warnings: sexual tension (like A LOT), inappropriate behaviour, protective!harry.

***

"You wanted to see me, Chef?"

You gave a light knock on the open door of Harry's office, trying to sound polite and professional. It had been a few weeks since you had that talk with Harry about keeping things strictly business between you two. During that time, he had been a perfect mentor - giving you advice and guidance without any flirting or suggestive comments.

His coaching had really helped improve your cooking skills as you soaked up all his knowledge and experience. You were grateful to have a normal working relationship again, focused solely on culinary training. And yet...you couldn't ignore the faint lingering tension between you, that subtle underlying charge.

Harry looked up from the notebooks on his desk, his eyes crinkling in a warm smile when he saw you. "Ah, there you are. Come on in, have a seat."

You sat down in one of the chairs across from him as Harry neatened up the loose papers into a stack. Up close, you couldn't help noticing how well-fitted his black button-down shirt was, or how his tousled hair looked very touchable.

Firmly reminding yourself this was just a professional meeting, you averted your eyes politely until Harry cleared his throat.

"So as you know, the big Martin gala fundraiser is coming up in a few weeks," he began, shuffling through some folders. "It's one of the biggest events of the year for underprivileged culinary education programs. I'll be preparing the featured dish for their live auction, and I'd love for you to assist me on it."

Your eyes went wide with surprise at this prestigious opportunity. The Martin gala was a hugely famous event in Chicago's culinary scene, attracting all the wealthiest and most notable diners. For an up-and-coming chef to collaborate on the centerpiece dish was an amazing honor and chance to get exposure.

"Wow, yes of course!" you replied enthusiastically. "I would be absolutely honored, Chef. Thank you for this incredible opportunity."

Harry's dimples deepened as he smiled approvingly. "Don't thank me yet. We'll be under a huge spotlight to deliver an amazing showstopper dish. I expect you to rise to the challenge."

You quickly nodded. "You can count on me to give it my absolute best effort. I'm ready to do whatever work is needed."

"Excellent," Harry said in a slightly lower, huskier tone. "That's exactly what I like to hear."

For a moment, his voice had a heated quality that hinted at other situations where your eagerness might be welcome. You ignored the shiver it sent through you, reminding yourself this was strictly business now between you two.

Harry seemed to realize he was skirting the line, as he abruptly straightened up and all hints of flirtation disappeared as he switched fully into mentor mode. "Right, well let me walk you through my basic vision so far..."

You leaned forward attentively as he outlined preliminary ideas for a highly ambitious and avant-garde dish blending molecular gastronomy techniques with classic French cuisine fundamentals. It was wildly cutting-edge, even for a showpiece event like the Martin gala. But the more details Harry provided, the more that same thrill of adrenaline rushed through you whenever presented with a new culinary challenge to conquer.

For the next hour, the two of you bounced ideas back and forth in that unique creative flow state that chefs share. Harry's presence was magnetic, but you refused to get distracted by more physical aspects - like the stretch of his biceps against his crisp sleeves, the hint of toned abs beneath his open collar, or the raspy timbre of his voice dipping into that lower register as he passionately discussed certain techniques.

And oh, his damn tattoos.

No, you sternly told yourself as the conversation began wrapping up. Those days of getting flustered around him were over. Harry had made it clear where you stood, and you fully accepted those boundaries. Anything else was just self-torture.

"...but of course, those are just preliminary thoughts," Harry was saying as he collected the scattered folders into a neat pile. "We'll have plenty of time to refine the details over the next couple weeks."

You nodded, filing away the mental notes you'd taken during the discussion. "Absolutely, Chef. Just let me know whatever you need for prep or testing different ideas to get a head start."

"Will do." With an air of finality, Harry gathered up the pile and rose from his seat. You quickly stood up as well, not wanting him to loom over you in the enclosed space. For a beat, you both hovered awkwardly, the air seeming to thicken between you.

"Well then," Harry said, making no move to step past you towards the door. "I'd say this calls for a drink to celebrate our new collaboration, wouldn't you agree?"

Before you could reply, he turned and went to a small antique cabinet tucked in an alcove you hadn't noticed before. With a practiced hand, Harry selected a heavy glass decanter and two tumblers, placing them on the cabinet and expertly twisting off the stopper.

"Let's go with Lagavulin," he mused aloud, carefully pouring two generous glasses of the amber scotch whisky. "A good Scottish whisky seems appropriate for the occasion."

"I really shouldn't, Chef," you said reflexively, already picturing your lightweight self getting sloppy and unprofessional after even a single drink.

But Harry just chuckled softly. "Loosen up a little. It's a celebration, after all."

He emphasized this by bringing one of the heavy tumblers over and pressing the cool glass into your hand. You frowned down at the coppery liquid, worrying your lower lip uncertainly. But before you could protest further, Harry gently clinked his glass against yours in a silent toast before taking a sizable sip.

The whisky's smoky, peaty aroma seemed to wrap around you intimately. Despite your hesitation, you couldn't help giving an appreciative inhale before taking a small, tentative sip yourself. Bold, layered flavors of vanilla, caramel, and charred oak underscored by an earthy smokiness burst over your tongue. You let out a soft sigh of indulgent pleasure at the decadent taste.

"Good, isn't it?" Harry's gravelly voice made you start slightly. He was watching you with amusement, whisky glass dangling casually from those large, handsome fingers. "It really hits you in the back of the throat, makes you slow down and savor it fully."

You suddenly realized the suggestive implication behind his phrasing and felt a flush of heat bloom across your face and chest. Harry watched the play of emotions flickering over your features with relish before taking another indulgent sip. This time, you noticed the way his full lips pursed delicately to drink, the tiny furrow of concentration between his brows as he savored the flavor before swallowing.

Unconsciously, your eyes tracked the mesmerizing flex of his throat as he swallowed, the hint of stubble grazing along his chiseled jawline. A twinge low in your abdomen accompanied the thought of feeling that scratchy burn of beard between your thighs, that talented mouth working magic elsewhere on your body.

Mortified, you shut down that wayward trail of thought through sheer willpower. Your cheeks grew even hotter as you realized Harry had caught you staring, his own gaze darkly amused.

"Easy there," he murmured huskily, stepping a bit deeper into your personal space. "This dish is a marathon, not a sprint. Best to learn to savor every indulgent morsel along the way."

With a pointed look and arched brow, Harry raised his whisky to those plump lips once more, holding your gaze as he placed the rim against that full lower lip and let out an obscenely gratifying groan of pure delight.

Moments after, the tension had subsided, but the flush and blush that had creeped up your cheeks wasn’t going away anytime soon–you were sure of that.

***

You tried to push aside the lingering thoughts about the “Celebration” that were now implaed into your mind, and the way tiny droplets of the drink remained on his lips till he licked them off with his tongue–

You wanted that tongue to be yours.

Shaking your head, you focused on prepping the ingredients for the evening service. The dinner rush would be starting soon and you needed to have everything ready. As you worked, you were vaguely aware of the dining room filling up with patrons being seated. The sounds and aromas of the bustling kitchen surrounded you in a familiar, comforting way.

You were so engrossed in your tasks that you didn't notice the man approach until he cleared his throat loudly. Looking up, you saw a smartly-dressed diner smiling at you in a way that made you instinctively uncomfortable.

"Well, hello there," he said in a syrupy tone. "I was just admiring the delicious-looking fare over here." He raked an obvious look up and down your body. "The menu selections have my mouth watering already."

You stiffened, recognizing the overly familiar leer. This wasn't the first time you'd dealt with an obnoxious patron hitting on you. Keeping your expression neutral, you replied in a polite but firm tone. "I'm afraid you'll need to return to the dining room, sir. The kitchen is off-limits to guests."

Rather than taking the hint, the man leaned nonchalantly against your prep station. "Don't be like that, sweetheart. I was just hoping you could suggest something...special for me to sample tonight." He punctuated this with an exaggerated wink.

Suppressing a grimace, you turned away to continue your work, hoping he would give up and leave. No such luck. The lech sidled closer until he was nearly pressed against you. "What do you say? I'd love for a tasty little thing like you to--"

"Sir, I'm going to have to ask you to leave the kitchen area immediately." Harry's firm baritone cut across the man's words like a whip crack.

You looked up in relief to see your boss standing with arms crossed, jaw clenched as he glared at the offending patron. Even from several feet away, you could sense the potent force of his displeasure rolling off him in waves.

The diner seemed to shrink slightly under Harry's censorious scowl. "Oh, uh, my apologies. I was just trying to get some personal recommendations--"

"The kitchen is off-limits and you're making my staff uncomfortable," Harry interrupted, his tone brooking no argument. "I won't ask again. Return to your table or you'll be asked to leave the premises."

Looking sufficiently cowed, the lech swiftly retreated with some mumbled apologies. You exhaled slowly, trying to dispel the anxiety brought on by the unpleasant encounter. Harry stepped closer, his expression softening as he looked you over with concern.

"You okay? That asshole didn't go too far, did he?"

You managed a faint smile, oddly touched by the protective edge in his voice. "I'm fine, Chef. Just another boorish customer thinking the uniform is a dinner invitation."

His jaw tightened again as he scowled in the direction the man had gone. "That type of behavior is completely unacceptable. You let me know right away if anyone hassles you like that again, understand?"

Nodding, you found yourself blinking rapidly against the unexpected prickle of grateful tears at having Harry firmly in your corner, despite the complicated dynamics between you lately.

For a long moment, he watched you carefully as if gauging your equilibrium. Then Harry surprised you by reaching out and briefly squeezing your shoulder in a reassuring gesture. The warmth of his large hand seeped through your uniform, leaving a tingly imprint even after he pulled away.

"I've got your back, [Y/N]. You focus on doing your job and let me deal with any assholes who get out of line."

The gruff tenderness in his words made your heart do a traitorous little flip in your chest. You nodded again, not trusting your voice enough to respond properly.

With one final pointed look, Harry turned and headed back out to his front-of-house duties. As you watched his broad-shouldered form disappear through the swinging doors of the kitchen, you felt a complicated tangle of gratitude, protectiveness, affection...and yes, a lingering undercurrent of attraction that you couldn't seem to fully extinguish despite your best efforts.

You spent the rest of the dinner service determinedly pushing aside any lingering thoughts about Harry or the earlier incident. Focusing fully on your work was the only way to get through these confusing emotions that had you all over the place..

The rhythm of prepping, plating, and coordinating with the other line cooks settled into a familiar, reassuring routine. The constant flurry of chopping, sautéing, and barked orders provided a sort of meditative escape from your muddled headspace.

By the time the last diner had been served and the kitchen was winding down for the night, you felt pleasantly drained in that satisfying way that comes from a job well done. As you began breaking down your station for cleaning, Harry emerged from his office looking satisfied.

"Excellent work tonight, everyone," he called out in that effortlessly commanding tone. "Front-of-house said the new salmon dish was a huge hit. We'll definitely want to keep that one on the seasonal menu."

A chorus of tired but pleased murmurs went around the kitchen at the praise. Harry's eyes found yours amidst the small crowd, holding your gaze a beat longer than strictly necessary before moving on to the other cooks. You tried not to read too much into it.

With the nightly pep talk concluded, Harry rolled up the sleeves of his crisp white chef's coat, joining everyone in the evening breakdown and cleaning duties. You watched surreptitiously as he expertly broke down one of the grill stations, muscles in his broad forearms flexing enticingly with each efficient movement.

Get a grip, you scolded yourself, quickly refocusing on scrubbing down your own prep area. This was exactly the kind of distracted, unprofessional behavior you were trying to avoid lately around Harry.

Despite your best efforts, however, you couldn't fully ignore him moving about the kitchen, checking in with each station to oversee their sanitation. At one point, he paused to examine some utensils that hadn't been properly cleaned, tsking in displeasure before batting them aside to be re-scrubbed.

"That's never going to meet inspection," he chided the sheepish-looking young line cook in his trademark gruff tone. "Do it again, and do it properly this time. We're not running a greasy spoon here."

As much as his uncompromising attitude could be intimidating, you also found it oddly...thrilling to witness Harry taking charge so authoritatively. Not to mention the visual of those powerful hands deftly at work was sending your thoughts in an unprofessional direction yet again.

Sternly redirecting your focus, you turned your back to give the area behind the grill station a thorough scrubbing. You were so engrossed that you nearly jumped out of your skin when Harry's low voice sounded directly in your ear.

"Everything looking good over here?"

You whirled around to find him looming directly behind you, near enough that you could smell the spicy notes of his subtle cologne mingling with the lingering kitchen aromas clinging to him. Up this close, you couldn't help noticing how the top buttons of his coat had come undone at some point, offering a teasing glimpse of the toned chest beneath.

Trying not to stare, you quickly averted your eyes as you nodded. "Y-yes, Chef. All clean on this side."

"Hmm." His assessing gaze slowly raked over your work before returning to your flushed face. The tiniest of smirks played about his lips as if he could read the direction of your thoughts.

"Well, then. Carry on," was all he said before turning and strolling unhurriedly back towards his office, burgundy cargo pants slung enticingly low on those lean hips.

You let out a shaky breath, mentally cursing how easily flustered you still became around this man, no matter how much you tried to enforce boundaries. Resolutely, you refocused on finishing your cleaning tasks, determined to get out of there before any more distracted lapses in professionalism.

By the time the kitchen had been scoured from top to bottom, you were one of the last few staffers remaining. Wearily peeling off your apron, you were just reaching for your bag when Harry reappeared, looking unhurried and relaxed now that the nightly duties were done.

"Heading out?" he asked as you approached, one thick eyebrow raised questioningly.

You stifled a yawn with the back of your hand. "Yeah, I'm beat. Gonna try and get some extra sleep before the morning prep shift tomorrow."

He made a noncommittal sound, falling into step beside you as you headed for the employee exit out back. For a few moments, you walked in silence, oddly aware of the warmth radiating off his body this close to yours.

When he finally spoke, it wasn't at all what you expected. "You did good with that asshole customer earlier."

Your steps faltered slightly at the praise before quickly recovering. "Oh...uh, thanks, Chef. You really didn't need to step in like that."

"The hell I didn't," he countered gruffly. There was an edge to his tone that made the tiny hairs at your nape prickle. "No one treats my staff like piece of meat, especially not in my own goddamn kitchen."

Harry shook his head in disgust at the very idea, causing a lock of mahogany hair to fall rakishly across his furrowed brow in a way that really shouldn't have been as distracting as it was.

Swallowing hard, you refocused on the matter at hand. "I've dealt with guys like that before. Just comes with the territory sometimes, y'know?"

"That doesn't make it acceptable," he insisted, mouth setting into a grim line. You found yourself unable to look away from the sharp angles of his frowning profile, chiseled jaw ticking faintly with irritation, that he tried to mask.

He fixed you with those intense pale eyes, all traces of humor gone. "No one - and I mean no one - gets to treat any of you with disrespect while I'm in charge around here. I won't stand for that shit under my roof."

The ferocity in his tone sent an involuntary shiver rippling through you, though from wariness or...something else entirely, you couldn't say. All you knew was the low, authoritative resonance of Harry's voice carried an unmistakable air of command that raised goosebumps along your arms.

Maybe it was the late hour, or the fact you were walking in such close proximity out of public view. Or hell, maybe it was just the sheer presence of this man who could flip between stern taskmaster and something rawer, more carnal in the blink of an eye.

Whatever it was, you felt that subtle spark between you ignite and suddenly, you desperately needed to be alone to process the yearning that flickered to life low in your belly. Before you could consider the impulse further, you were blurting out the first excuse that came to mind.

"Well, thanks again for that. And for the whole mentorship thing too. I, uh...I actually have some errands to run, so I'll just catch you tomorrow morning, 'kay?"

You didn't even give Harry a chance to respond before ducking through the exit, muscles taut with confused tension. As the cool night enveloped you, you drew a deep, shuddering breath in an effort to steady yourself.

Whatever weird atmospheric flux had momentarily enveloped you back there was too dangerous, too distracting from the tenuous balance you and Harry had only just reestablished. No, it was better to put some space between you before things got muddied again.

With a fierceness born of sheer force of will, you wrestled your turbulent, wandering thoughts back under control. You were a professional, with goals to work towards. Getting pulled into Harry's electrifying orbit again would only derail you.

Still, as you hurried to your car, his shape-shifting countenance kept flashing unbidden across your memory - the dazzling smile, the brooding intensity, the simmering promise of authority barely restrained. All of it provided an infuriatingly potent combination that had your body humming with repressed longing despite yourself.

This was going to take more effort than you'd anticipated.

***

The next couple of weeks passed in a blur of grueling practice runs and preparation for the Martin gala. You and Harry spent nearly every waking hour in the kitchen, iterating endlessly on his showpiece dish concept.

With the prestigious event date rapidly approaching, any lingering awkwardness or tension between you had been shifted firmly into the background. The shared urgency of perfecting this culinary masterpiece became an all-consuming focus that left little room for anything else.

Still, that didn't stop you from noticing...things.

Like how the sleeves of Harry's whites had an endearing tendency to get shoved up his forearms in a way that displayed those tanned, sinewy muscles to distracting effect as he worked. You definitely didn't linger over the sight of his strong hands deftly wielding a knife, making precise, practiced cuts. And you absolutely did not imagine those dexterous fingers trailing across your skin instead of the cutting board.

At least, that's what you sternly told yourself in an ongoing effort to maintain focus.

For his part, Harry was all business during these preparation sessions - issuing clipped instructions, evaluating ingredients with a critical eye, pushing both of you relentlessly to get every component just right. Only rarely did you catch hints of something more underneath that professional veneer.

Like the time you were bent over a burner, carefully spooning out the orbs of flavored olive oil onto the waiting plate. Harry stepped up behind you to examine your work, the warmth of his body radiating against your back. As he leaned in closer to inspect the delicate orbs, his low murmur caressed the fine hairs at your nape in a way that made you shiver.

"That's it...go nice and slow with a deft touch," he rumbled in that raspy timbre that never failed to send tingles shooting straight to your core.

Heart pounding, you risked a sidelong glance to find his pale eyes already locked on yours, glittering with an intensity that contrasted sharply with his deceptively neutral expression. A charged moment stretched between you as that underlying spark you'd been determinedly ignoring flared, sudden and molten.

Just when you thought you might spontaneously combust, Harry blinked and cleared his throat brusquely. "Carry on, then," he instructed in his normal crisp tone before turning away to focus on another component.

You stood motionless for several heartbeats, fingers clenched around the spoon, skin flushed and tingling in equal measures of arousal and disbelief. Did that really just happen or had the endless hours in the kitchen started affecting your mind?

Too skittish to ponder it further, you dove back into your tasks with even more single-minded focus, the uneasy moment shelved and locked away tight. No matter what fleeting tension arose in isolated pockets, you couldn't afford to unpack it right now - not with the enormity of what was at stake.

The days ticked down in a relentless march until finally, you and Harry stood in the solitude of his spartan office the night before the big event, taking a breather from your marathon final prep session.

An ungodly number of mise en place containers filled every available surface, each holding fussed-over components of the highly elaborate and conceptual dish that would make its debut tomorrow. Harry had pushed you both to your physical and creative limits, drilling the execution repeatedly until he was satisfied you could plate it flawlessly under the anticipated scrutiny.

Now, having quality-checked and prepped every last possible element, there was nothing further to do except rest up and bring your sharpest mental game tomorrow. Harry seemed to deflate slightly as the backdrop of mounting pressure decreased for the first time in weeks.

Propping his hip against the desk with studied nonchalance, he quirked one eyebrow in a sidelong glance. "You ready for this?"

Despite your weariness, you felt that familiar thrill of adrenaline stir at those simple words - as well as a contradictory quiver of nerves. This event was a make-or-break opportunity of the highest magnitude, especially for someone like you just starting out. Either you nailed your responsibilities tomorrow, or it all came crashing down in front of Chicago's most elite gourmands.

Shoving aside the sudden flutters of doubt, you met Harry's inscrutable gaze head-on, straightening your spine. "You know I am. We've put in the work, and this dish is gonna blow them all away."

A tiny smirk tugged at the corner of his sculpted mouth as he studied you appraisingly. "That's what I like to hear. Just remember - all the technique practice in the world won't mean a thing if you panic out there."

The subtle warning made you bristle defensively, never one to back down from a challenge. "I'm not going to panic," you scoffed. "I eat massive amounts of public pressure like this for breakfast."

Harry's eyes danced with amusement, and not for the first time, it struck you how effortlessly he could switch between imposing and playful. "Is that so?" he drawled easily. "In that case, would you care to make things a bit more interesting?"

Before you could respond, Harry kicked off from the desk in one sinuous motion to prowl closer. Despite your weariness, you felt your heart rate kick up several notches as he invaded your personal space, long body coiled with a loose, predatory grace.

"Let's say we raise the stakes a little," he proposed in a tone of studied nonchalance that was completely belied by the heated glint in his eyes boring into yours. "If you can prove you've got the chops to keep a cool head under fire tomorrow, I'll take you out afterwards to celebrate. Just you and me, anywhere you want to go."

Your mouth went instantly dry at the implications behind his offer. Were those...the unmistakable undertones of flirtation coloring his invitation? After the weeks of him keeping things strictly professional between you, the sudden shift was dizzying - and left you dangerously intrigued.

"And what if I choke?" you heard yourself countering recklessly before you could reconsider. "What do you get out of it then?"

His answering smile was pure blistering sin. "Oh, sweetheart. If that happens...I get to take you out too - but somewhere a bit more private."

Harry paused to let the suggestive proposition linger, backing it up with a slow, heated raking of his pale eyes over your body that left zero doubt as to his implication. Heat bloomed furiously across your cheeks as forbidden images flooded your mind unbidden - flashes of tangled limbs, straining muscle, sweaty exertion of a far different sort...

Then, just like that, the provoking spell was broken. Rocking back on his heels, Harry shrugged one broad shoulder in an easy, dismissive gesture. "But that's not going to happen, is it? You've got all the skills, you've put in the time - no reason to buckle tomorrow."

He threw one final weighted glance in your direction before pivoting on his heel towards the door. "Get some rest. I'll see you at the venue early to do our final walkthrough before we get this show on the road."

And with that parting comment, Harry strode casually out, leaving you rooted there in dumbfounded silence. What the hell had just happened? One moment, you'd merely been steeling yourselves for tomorrow's high stakes challenge - and then suddenly he was issuing some bizarrely flirtatious...proposition.

Or was that really what it was? As you stood there chasing replays of his words, his tone, his body language - the whole previous interaction kept taking on a slinkier, more salacious cast. Like maybe your presence of mind was slipping already, causing you to read into things that weren't really there.

No...no, you decided as you hefted your bag, determined to put it all out of your head for now. Harry was just his usual aggravating self, trying to rile you by dangling some imagined reward or punishment to keep you on your toes before the big event. This whole...suggestive semiflirtation thing was just the product of your own exhausted mind playing tricks.

Firmly shoving aside all unsettling thoughts, you focused on the immediate challenge awaiting tomorrow. You would plate Harry's showpiece dish to absolute perfection, prove yourself under the brightest lights, and decisively seize this career-making opportunity.

Everything else could be dealt with later.

♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡~~~♡

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