#Cyclin

[1337wtfomgbbq]

[9d92hd9ge]

[detragefietser]

tropical dream

[life-spire]

@ fhmi29

[joanofarc]

cyclin' round town!, poetry slam bam thank u mam (2024).

look left and look right for thoughts that just might collide into you i fell and i grew

[myths-of-fantasy]

I should be working on my other days

I am actually integrating magic more into The Black Wolf Capers.

Anyway VILE was now founded by a cult that worshipped a god, Greed who would offer his followers endless and eternal riches and is in constant conflict with the followers of Envy. Carmen was meant to be a vessel for Greed, merging with her soul to grant him physical form so he could better influence the world, but Dexter Wolfe tried to run off with her. It didn't work obviously, but eventually Carmen escaped before the transfer could happen.

Now VILE is chasing her around the world, both bc she's destroying their organization and also is preventing them from releasing Greed.

[myrawjcsmicasereports]

 Inhibition of EIF4E Downregulates VEGFA and CCND1 Expression to Suppress Ovarian Cancer Tumor Progression by Jing Wang in Journal of Clinical Case Reports Medical Images and Health Sciences

Abstract

This study investigates the role of EIF4E in ovarian cancer and its influence on the expression of VEGFA and CCND1. Differential expression analysis of VEGFA, CCND1, and EIF4E was conducted using SKOV3 cells in ovarian cancer patients and controls. Correlations between EIF4E and VEGFA/CCND1 were assessed, and three-dimensional cell culture experiments were performed. Comparisons of EIF4E, VEGFA, and CCND1 mRNA and protein expression between the EIF4E inhibitor 4EGI-1-treated group and controls were carried out through RT-PCR and Western blot. Our findings demonstrate elevated expression of EIF4E, VEGFA, and CCND1 in ovarian cancer patients, with positive correlations. The inhibition of EIF4E by 4EGI-1 led to decreased SKOV3 cell clustering and reduced mRNA and protein levels of VEGFA and CCND1. These results suggest that EIF4E plays a crucial role in ovarian cancer and its inhibition may modulate VEGFA and CCND1 expression, underscoring EIF4E as a potential therapeutic target for ovarian cancer treatment.

Keywords: Ovarian cancer; Eukaryotic translation initiation factor 4E; Vascular endothelial growth factor A; Cyclin D1

Introduction

Ovarian cancer ranks high among gynecological malignancies in terms of mortality, necessitating innovative therapeutic strategies [1]. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis, influencing endothelial cell proliferation, migration, vascular permeability, and apoptosis regulation [2, 3]. While anti-VEGF therapies are prominent in malignancy treatment [4], the significance of cyclin D1 (CCND1) amplification in cancers, including ovarian, cannot be overlooked, as it disrupts the cell cycle, fostering tumorigenesis [5, 6]. Eukaryotic translation initiation factor 4E (EIF4E), central to translation initiation, correlates with poor prognoses in various cancers due to its dysregulated expression and activation, particularly in driving translation of growth-promoting genes like VEGF [7, 8]. Remarkably, elevated EIF4E protein levels have been observed in ovarian cancer tissue, suggesting a potential role in enhancing CCND1 translation, thereby facilitating cell cycle progression and proliferation [9]. Hence, a novel conjecture emerges: by modulating EIF4E expression, a dual impact on VEGF and CCND1 expression might be achieved. This approach introduces an innovative perspective to impede the onset and progression of ovarian cancer, distinct from existing literature, and potentially offering a unique therapeutic avenue.

Materials and Methods

Cell Culture

Human ovarian serous carcinoma cell line SKOV3 (obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was cultured in DMEM medium containing 10% fetal bovine serum. Cells were maintained at 37°C with 5% CO2 in a cell culture incubator and subcultured every 2-3 days.

Three-Dimensional Spheroid Culture

SKOV3 cells were prepared as single-cell suspensions and adjusted to a concentration of 5×10^5 cells/mL. A volume of 0.5 mL of single-cell suspension was added to Corning Ultra-Low Attachment 24-well microplates and cultured at 37°C with 5% CO2 for 24 hours. Subsequently, 0.5 mL of culture medium or 0.5 mL of EIF4E inhibitor 4EGI-1 (Selleck, 40 μM) was added. After 48 hours, images were captured randomly from five different fields—upper, lower, left, right, and center—using an inverted phase-contrast microscope. The experiment was repeated three times.

GEPIA Online Analysis

The GEPIA online analysis tool (http://gepia.cancer-pku.cn/index.html) was utilized to assess the expression of VEGFA, CCND1, and EIF4E in ovarian cancer tumor samples from TCGA and normal samples from GTEx. Additionally, Pearson correlation coefficient analysis was employed to determine the correlation between VEGF and CCND1 with EIF4E.

RT-PCR

RT-PCR was employed to assess the mRNA expression levels of EIF4E, VEGF, and CCND1 in treatment and control group samples. Total RNA was extracted using the RNA extraction kit from Vazyme, followed by reverse transcription to obtain cDNA using their reverse transcription kit. Amplification was carried out using SYBR qPCR Master Mix as per the recommended conditions from Vazyme. GAPDH was used as an internal reference, and the primer sequences for PCR are shown in Table 1.

Amplification was carried out under the following conditions: an initial denaturation step at 95°C for 60 seconds, followed by cycling conditions of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, repeated for a total of 40 cycles. Melting curves were determined under the corresponding conditions. Each sample was subjected to triplicate experiments. The reference gene GAPDH was used for normalization. The relative expression levels of the target genes were calculated using the 2-ΔΔCt method.

Western Blot

Western Blot technique was employed to assess the protein expression levels of EIF4E, VEGF, and CCND1 in the treatment and control groups. Initially, cell samples collected using RIPA lysis buffer were lysed, and the total protein concentration was determined using the BCA assay kit (Shanghai Biyuntian Biotechnology, Product No.: P0012S). Based on the detected concentration, 20 μg of total protein was loaded per well. Electrophoresis was carried out using 5% stacking gel and 10% separating gel. Subsequently, the following primary antibodies were used for immune reactions: rabbit anti-human polyclonal antibody against phospho-EIF4E (Beijing Boao Sen Biotechnology, Product No.: bs-2446R, dilution 1:1000), mouse anti-human monoclonal antibody against EIF4E (Wuhan Sanying Biotechnology, Product No.: 66655-1-Ig, dilution 1:5000), mouse anti-human monoclonal antibody against VEGFA (Wuhan Sanying Biotechnology, Product No.: 66828-1-Ig, dilution 1:1000), mouse anti-human monoclonal antibody against CCND1 (Wuhan Sanying Biotechnology, Product No.: 60186-1-Ig, dilution 1:5000), and mouse anti-human monoclonal antibody against GAPDH (Shanghai Biyuntian Biotechnology, Product No.: AF0006, dilution 1:1000). Subsequently, secondary antibodies conjugated with horseradish peroxidase (Shanghai Biyuntian Biotechnology, Product No.: A0216, dilution 1:1000) were used for immune reactions. Finally, super-sensitive ECL chemiluminescence reagent (Shanghai Biyuntian Biotechnology, Product No.: P0018S) was employed for visualization, and the ChemiDocTM Imaging System (Bio-Rad Laboratories, USA) was used for image analysis.

Statistical Analysis

GraphPad software was used for statistical analysis. Data were presented as (x ± s) and analyzed using the t-test for quantitative data. Pearson correlation analysis was performed for assessing correlations. A significance level of P < 0.05 was considered statistically significant.

Results

3D Cell Culture of SKOV3 Cells and Inhibitory Effect of 4EGI-1 on Aggregation

In this experiment, SKOV3 cells were subjected to 3D cell culture, and the impact of the EIF4E inhibitor 4EGI-1 on ovarian cancer cell aggregation was investigated. As depicted in Figure 1, compared to the control group (Figure 1A), the diameter of the SKOV3 cell spheres significantly decreased in the treatment group (Figure 1B) when exposed to 4EGI-1 under identical culture conditions. This observation indicates that inhibiting EIF4E expression effectively suppresses tumor aggregation.

Expression and Correlation Analysis of VEGFA, CCND1, and EIF4E in Ovarian Cancer Samples

To investigate the expression of VEGFA, CCND1, and EIF4E in ovarian cancer, we utilized the GEPIA online analysis tool and employed the Pearson correlation analysis method to compare expression differences between tumor and normal groups. As depicted in Figures 2A-C, the results indicate significantly elevated expression levels of VEGFA, CCND1, and EIF4E in the tumor group compared to the normal control group. Notably, the expression differences of VEGFA and CCND1 were statistically significant (p < 0.05). Furthermore, the correlation analysis revealed a positive correlation between VEGFA and CCND1 with EIF4E (Figures 2D-E), and this correlation exhibited significant statistical differences (p < 0.001). These findings suggest a potential pivotal role of VEGFA, CCND1, and EIF4E in the initiation and progression of ovarian cancer, indicating the presence of intricate interrelationships among them.

EIF4E, VEGFA, and CCND1 mRNA Expression in SKOV3 Cells

To investigate the function of EIF4E in SKOV3 cells, we conducted RT-PCR experiments comparing EIF4E inhibition group with the control group. As illustrated in Figure 3, treatment with 4EGI-1 significantly reduced EIF4E expression (0.58±0.09 vs. control, p < 0.01). Concurrently, mRNA expression of VEGFA (0.76±0.15 vs. control, p < 0.05) and CCND1 (0.81±0.11 vs. control, p < 0.05) also displayed a substantial decrease. These findings underscore the significant impact of EIF4E inhibition on the expression of VEGFA and CCND1, indicating statistically significant differences.

Protein Expression Profiles in SKOV3 Cells with EIF4E Inhibition and Control Group

Protein expression of EIF4E, VEGFA, and CCND1 was assessed using Western Blot in the 4EGI-1 treatment group and the control group. As presented in Figure 4, the expression of p-EIF4E was significantly lower in the 4EGI-1 treatment group compared to the control group (0.33±0.14 vs. control, p < 0.001). Simultaneously, the expression of VEGFA (0.53±0.18 vs. control, p < 0.01) and CCND1 (0.44±0.16 vs. control, p < 0.001) in the 4EGI-1 treatment group exhibited a marked reduction compared to the control group.

Discussion

EIF4E is a post-transcriptional modification factor that plays a pivotal role in protein synthesis. Recent studies have underscored its critical involvement in various cancers [10]. In the context of ovarian cancer research, elevated EIF4E expression has been observed in late-stage ovarian cancer tissues, with low EIF4E expression correlating to higher survival rates [9]. Suppression of EIF4E expression or function has been shown to inhibit ovarian cancer cell proliferation, invasion, and promote apoptosis. Various compounds and drugs that inhibit EIF4E have been identified, rendering them potential candidates for ovarian cancer treatment [11]. Based on the progressing understanding of EIF4E's role in ovarian cancer, inhibiting EIF4E has emerged as a novel therapeutic avenue for the disease. 4EGI-1, a cap-dependent translation small molecule inhibitor, has been suggested to disrupt the formation of the eIF4E complex [12]. In this study, our analysis of public databases revealed elevated EIF4E expression in ovarian cancer patients compared to normal controls. Furthermore, through treatment with 4EGI-1 in the SKOV3 ovarian cancer cell line, we observed a capacity for 4EGI-1 to inhibit SKOV3 cell spheroid formation. Concurrently, results from PCR and Western Blot analyses demonstrated effective EIF4E inhibition by 4EGI-1. Collectively, 4EGI-1 effectively suppresses EIF4E expression and may exert its effects on ovarian cancer therapy by modulating EIF4E.

Vascular Endothelial Growth Factor (VEGF) is a protein that stimulates angiogenesis and increases vascular permeability, playing a crucial role in tumor growth and metastasis [13]. In ovarian cancer, excessive release of VEGF by tumor cells leads to increased angiogenesis, forming a new vascular network to provide nutrients and oxygen to tumor cells. The formation of new blood vessels enables tumor growth, proliferation, and facilitates tumor cell dissemination into the bloodstream, contributing to distant metastasis [14]. As a significant member of the VEGF family, VEGFA has been extensively studied, and it has been reported that VEGFA expression is notably higher in ovarian cancer tumors [15], consistent with our public database analysis. Furthermore, elevated EIF4E levels have been associated with increased malignant tumor VEGF mRNA translation [16]. Through the use of the EIF4E inhibitor 4EGI-1 in ovarian cancer cell lines, we observed a downregulation in both mRNA and protein expression levels of VEGFA. This suggests that EIF4E inhibition might affect ovarian cancer cell angiogenesis capability through downregulation of VEGF expression.

Cyclin D1 (CCND1) is a cell cycle regulatory protein that participates in controlling cell entry into the S phase and the cell division process. In ovarian cancer, overexpression of CCND1 is associated with increased tumor proliferation activity and poor prognosis [17]. Elevated CCND1 levels promote cell cycle progression, leading to uncontrolled cell proliferation [18]. Additionally, CCND1 can activate cell cycle-related signaling pathways, promoting cancer cell growth and invasion capabilities [19]. Studies have shown that CCND1 gene expression is significantly higher in ovarian cancer tissues compared to normal ovarian tissues [20], potentially promoting proliferation and cell cycle progression through enhanced cyclin D1 translation [9]. Our public database analysis results confirm these observations. Furthermore, treatment with the EIF4E inhibitor 4EGI-1 in ovarian cancer cell lines resulted in varying degrees of downregulation in CCND1 mRNA and protein levels. This indicates that EIF4E inhibition might affect ovarian cancer cell proliferation and cell cycle progression through regulation of CCND1 expression.

In conclusion, overexpression of EIF4E appears to be closely associated with the clinical and pathological characteristics of ovarian cancer patients. In various tumors, EIF4E is significantly correlated with VEGF and cyclin D1, suggesting its role in the regulation of protein translation related to angiogenesis and growth [9, 21]. The correlation analysis results in our study further confirmed the positive correlation among EIF4E, VEGFA, and CCND1 in ovarian cancer. Simultaneous inhibition of EIF4E also led to downregulation of VEGFA and CCND1 expression, validating their interconnectedness. Thus, targeted therapy against EIF4E may prove to be an effective strategy for treating ovarian cancer. However, further research and clinical trials are necessary to assess the safety and efficacy of targeted EIF4E therapy, offering more effective treatment options for ovarian cancer patients.

Acknowledgments:

Funding: This study was supported by the Joint Project of Southwest Medical University and the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University (Grant No. 2020XYLH-043).

Conflict of Interest: The authors declare no conflicts of interest.

[starstruckkittenglitter]

[pestercide]

Temporarily makin my pinned post smthn I drew that I'm proud of because I'm allowed to as a treat :3c

[summerlycoris]

Hell yeah! This is how you know it's summertime! CYCLONE SEASON M8'S

[astaroth1357]

It Must Be Done (Chat Spoilers for NB):

MC: ..... *sets down their phone*

MC: I know what I must do.

*one hour later in the House of Lamentation, Mammon walks up the stairs to go get Satan for dinner, just to find their attendant already waiting outside of the fourthborn's door... putting on a bike helmet?*

Mammon: *walks up beside them, frowning* Eh? MC? Is that a helmet?

MC: *locks in the straps and makes sure they're secure*

MC: Yep.

Mammon: .... Okaaaaay? Are ya goin' cyclin' or....?

MC: Nope.

*the MC brings their hand up and bangs it against Satan's door before jogging to the far wall. They take just a few seconds to stop, flip around, and get into a running stance*

Mammon: What the hell are ya-??

*Satan swings open his door with a look of growing annoyance, prompting MC to start sprinting straight at him*

Satan: Huh??

*The blonde barely has time to process to what's happening before the MC goes airborne and careens head first into his chest, rocketing them both back into his bedroom*

Mammon: WHAT THE FUCK ARE YOU DOING!?!

*Mammon runs through the doorway and finds Satan sprawled out flat on his back with MC half on top of him, dazed. MC sits up and rubs their neck while he remains still*

MC: Ooooww... that hurt.... I told you it was dangerous.

Mammon: MC, why did you do that?! Are you insane?!? Get outta there he's gonna-!! He's...! gonna.....?

*Mammon watches in disbelief as Satan not only stays still, but slowly gets redder and redder from the tips of his ears down to his cheeks. Eventually he has to resort to masking his face behind his hands entirely while MC triumphantly grins*

MC: Was that what you wanted~?

Satan: *finally sits up, unable to look them in eye*

Satan: Please don't do that again...

Mammon: *watches the scene, getting more confused by the second* .... Y'all are weird. Come downstairs and eat already!

[vhstown]

ily nana but unfortunately they are hashtag boring AF! (i cba)

i cannot take myself seriously why is my cyclin dependent kinase inhibitor research next to ain't no love part 5 on my google docs 😭😭😭😭

[saintshigaraki]

sorry if u dont answer these kinds of questions but ive been slowly eatin ur dabi thoughts like crumbs off the floor and i am curious as to what makes you think he'd be clingy/possessive in a relationship??

cells are able to enter the mitosis phase and divide because of MPF, mitosis promoting factor. they're cyclin dependent kinases. but it takes a lot for MPF to become active, because the cyclin attached is inhibited by a phosphate group. eventually, though, a protein comes along and removes the phosphate which creates this insane sort of positive feedback loop and suddenly MPF is extremely active. touya in a romantic relationship sort of reminds me of that insane positive feedback loop.

i think you guys do honestly start out as....fuck buddies for a lack of a better word. but it probably all started because you were kind to him. you let him crash at your place because he reminded you of a drowned kitten, which, dabi will tell you later, was so incredibly fucking stupid of you. but it's all these little kindnesses you show him that build up to this.....obsessive, possessive behavior. dabi's already primed for this because he has some truly insane attachment and abandonment issues. one moment he's relatively normal and the next.....it's the same sort of on/off reaction seen in cells with mitosis, except, unlike in healthy cells, he never turns off once on. more reminiscent of a cancer cell. he just...doesn't want to lose you. and i dont think he has a very healthy secure view of himself. he imagines you'd be better off with practically anyone else. but he doesn't want you to be with anyone else. so he'll do whatever it takes to keep you with him. within arms reach. always.

[detragefietser]

[allthedesiredusernamesaretaken]

Born today 60 years ago - rockin' and rollin' and cyclin'

📷 Mark Seliger

[jokertrap-ran]

[Gakuen K] Kusanagi Izumo Route: White Day’s return gift Translation

*Translator’s note : MC’s name shall remain as my normal (水嶋ラン) *Gakuen K Masterlist / Gakuen K Mobile Masterlist *Spoiler FREE : Translations under cut !

Izumo: Are you really sure this is enough for your white day gift?

Ran: Yup. This plushie’s really cute!

Ran: Its eyes look just like yours!

Izumo: Really? Aw, now I’m flustered,

Izumo: I was actually plannin’ on gettin’ accessories for you or somethin’ like that…

Ran: Would that be better?

Izumo: No, I think it’s cute, and gettin’ a plushie really fits your character…

Izumo: I guess that marks the end of our shoppin’ session. How ‘bout we go for a drive?

Ran: A drive?

Izumo: Yeah. It’s cold out, but the weather’s nice, so let’s go somewhere further away.

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Izumo: —We’re here. A reward for sitting through the short drive.

Ran: Whoa…

Izumo: There are some things out there that are hard to see, even if they’re just right under your nose. We’re just a lil’ way out from the city, but you can see such a wide expanse of the starry sky. Amazin’ don’t you think?

Izumo: I came here a couple of years ago with Mikoto, cyclin’ ‘round the island on a bike. We chanced upon this place after decidin’ to do somethin’ stupid or another.

Izumo: You can get here quickly by car, but it’s a tad difficult to walk or cycle here. I often come here whenever I need to think.

Izumo: Lookin’ at the vast starry sky up there, my own worries feel like a speck of dust in comparison.

Izumo: And the sound of the waves are also very soothin’…

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Ran: Kusanagi-san…

Izumo: But, well, now I have you. And you are like the soothin’ balm in my life, so I don’t have much use for the starry skies now, do I?

Izumo: You’ll get cold if you stay that far away. C’mere.

Ran: Okay.

Izumo: …Closer. Are you tryin’ to make space?

Ran: I’m not… It’s just embarrassing.

Izumo: What’s there to be embarrassed about? We’re the only ones here.

Izumo: No one will see… even if I kiss you here.

Ran: Kusanagi-san…

Izumo: What is it?

Ran: It’s… nothing.

Izumo: Haha, what’s with that? If you can’t find the right words, then shh.

Izumo: If you can’t speak… Well, there are other ways to communicate, no?

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Izumo: I drove here as quickly as I could, but it looks like we've failed to make it in time. Can you open it..?

Ran: No… I can't open it since it's past curfew.

Izumo: I see. Looks like it's time to pull out my trump card.

Ran: What trump card?

Izumo: Take my hand and hold on tight.

Izumo: And here we are. You're welcome, princess.

Ran: That's gotta be my first time entering through the balcony…

Izumo: Really? I used to get into my dorm through the balcony more often than not when I was in high school, puttin’ the door to shame.

Izumo: Oh, right. Do remember to lock your balcony door. Else, what are you gonna do if an intruder comes in?

Ran: Like you?

Izumo: Yup, like m— wait, no!

Izumo: So this is your room. It looked totally different back in my day.

Ran: Oh, oops. Wait here, I’ll go turn on the lights. Would you like something to drink?

Izumo: Nah, I don’t plan on stayin’ long. Plus… It’d be bad if someone caught me here.

Ran: Aw, that’s a pity. I wanted to stay with you a little longer…

Izumo: Do you really want that?

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Ran: Kusanagi-san…?

Izumo: What a coincidence, I am of the same mind as well. I… would stay with you till the sun rises if I could.

Izumo: While hearin’ your voice, and stayin’ awake till the sun rises.

Izumo: What will you do if I plead for you to allow me to do so?

Ran: I…

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Izumo: Haha… 0 points. When that happens, you’ve gotta turn them down at once without hesitation.

Izumo: I’m gonna leave before someone else, or heaven forbid, the dormitory supervisor finds out I’m here. Sorry, but this is goodbye for today.

Ran: Kusanagi-san…

»» ━━━━━━━ ∘◦♔◦∘ ━━━━━━━ ««

Next Scene: Question