Biotechnology and Applied Biochemistry

Human papillomavirus‐16 E6‐positive cervical cancer attenuated by potent 2‐(4‐biphenylyl)‐N‐(1‐ethyl‐4‐piperidinyl) acetamide second‐generation analogs with improved binding affinity

AbstractHuman papillomavirus (HPV) infection, particularly HPV16, is a major contributor to the development of cervical cancer. Given the urgent need for novel therapeutic strategies targeting HPV‐associated cancers, this study focuses on characterizing second‐generation analogs of a lead compound, as a potential inhibitor of HPV16‐E6. Protein–ligand docking, Gibbs binding free energy estimation, and molecular dynamics simulations were conducted. HPV16‐infected SiHa and CaSki cell lines were used. MTT (3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide) assay for proliferation and flow cytometry for target inhibition and apoptosis were employed. Computational and cell proliferation analyses revealed that modifications to E6‐855, particularly in the piperidinyl group, enhanced binding affinities against HPV16‐E6, with E6‐272 demonstrating superior binding properties. Molecular dynamics simulations confirmed the stable binding of E6‐272 to HPV16‐E6, supported by favorable binding energy estimates. E6‐272 inhibited the proliferation of SiHa and CaSki cells with GI50 values of 32.56 and 62.09 nM, respectively. The compound reduced HPV16‐E6‐positive population, while inducing the early and late phase apoptosis in these cells. Structural alterations at the piperidinyl group of E6‐855 identified E6‐272 as a promising inhibitor of HPV16‐E6 with improved efficacy against HPV16‐E6. Further experimental validation of E6‐272 and its analogs warrant to advance its clinical utility in combating HPV‐associated cancers.

Correlation between plasma PSGL‐1 and FIGO stage, tumor metastasis, and survival in epithelial ovarian cancer

AbstractPlasma circulating P‐selectin glycoprotein ligand‐1 (PSGL‐1) levels and its clinical correlation in patients with epithelial ovarian cancer (EOC) are unknown. The study determined plasma PSGL‐1 levels in EOC patients and investigated its relationship with clinicopathological factors and prognosis. Plasma PSGL‐1 levels were measured using ELISA in 69 patients with EOC, 34 patients with benign ovarian cystadenoma, and 36 healthy controls. Subsequently, the relationship between PSGL‐1 levels and clinicopathological characteristics of patients, as well as the prognosis of EOC patients, was examined. Additionally, the specificity and sensitivity of plasma PSGL‐1 were assessed through ROC curve analysis. Plasma PSGL‐1 was upregulated in EOC patients compared with healthy subjects and/or patients with benign ovarian cystadenoma (p < 0.01). Elevated levels of PSGL‐1 in the plasma were positively associated with advanced FIGO stage (p < 0.001), tumor size (p = 0.001), tumor metastasis (p = 0.036), and tumor recurrence (p = 0.013), while was negatively correlated with residual tumor size (p < 0.001). Kaplan–Meier survival analysis showed that high plasma PSGL‐1 levels were associated with progression‐free survival (p = 0.0345). In univariate and multivariate Cox regression analyses, PSGL‐1 (HR = 1.456, p = 0.009) was an independent prognostic marker. Plasma PSGL‐1 levels distinguished EOC patients and healthy individuals (AUC = 0.905), patients at late and early FIGO stages (AUC = 0.886), and metastatic and non‐metastatic EOC (AUC = 0.722). The expression of plasma PSGL‐1 is significantly increased in patients with EOC, serving as a reliable biomarker to differentiate between healthy individuals and those with EOC. Furthermore, PSGL‐1 in patients is correlated with prognostic indicators, such as advanced FIGO stage, tumor lymph node metastasis, and progression‐free survival.

Repurposing of quinoline alkaloids identifies their ability to enhance doxorubicin‐induced sub‐G0/G1 phase cell cycle arrest and apoptosis in cervical and hepatocellular carcinoma cells

AbstractThe ability of quinoline alkaloids (cinchonine, cinchonidine, quinine, and quinidine) to sensitize different human cancer cell lines to doxorubicin (DOX)‐induced cell death was evaluated. Cell viability was analyzed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and the alkaloids ability to enhance DOX‐induced apoptosis was explored using Western blotting analysis. Also, flow cytometry was applied to analyze cell fractions in the different cell cycle phases. All alkaloids showed a significant enhancement of DOX‐induced cell death in HeLa and HepG2 cell lines. The chemosensitizing activity of the quinoline alkaloids was attributed to the induction of apoptosis as indicated by splitting of caspase‐3 and its substrate poly (ADP‐ribose) polymerase (PARP). In addition, there was an increase in the cell fractions in sub‐G0/G1 phase in case of DOX combination with the alkaloids. This study proves the ability of the quinoline alkaloids to enhance DOX‐induced apoptotic cell death in human cervical and hepatocellular carcinoma cells.

Glycogen synthase kinase‐3β inactivation promotes cervical cancer progression, invasion, and drug resistance

AbstractHuman papillomavirus (HPV) infection‐dependent cervical cancer is one of the most common gynecological cancers and often becomes aggressive, with rapid proliferation, invasion/migration, and drug resistance. Here, 135 fresh human cervical squamous cell carcinoma (CSCC) tissue specimens, comprising 21 adjacent normal (AN), 30 cervical intraepithelial neoplasia (CIN1–3), 45 CSCC, and 39 drugs (chemo‐radiation)‐resistant cervical tumor (DRCT) tissues were included. HPV‐positive (HeLa, SiHa), HPV‐negative (C33A), and cisplatin‐resistant (CisR–HeLa/–SiHa/–C33A) cell lines were used for in vitro studies. HPV16/18 oncoproteins E6/E7, pERK1/2, and glycogen synthase kinase‐3 (GSK3) and the matrix metalloproteinases (MMPs) MMP‐9/‐2 were assessed using immunohistochemistry, WB, and gelatin zymography. HPV16/18 infection was observed in 16.7% of the CIN1‐3, 77.8% of the CSCC, and 89.7% of DRCT samples. Total and inactive GSK3β expressions were associated with overall CSCC progression (p = 0.039 and p = 0.024, respectively) and chemoresistance (p = 0.004 and p = 0.014, respectively). Positive correlations were observed, between the expression of E6 and pGSK3β expression (p = 0.013); E6 and CSCC progression (p < 0.0001)/drug resistance (p = 0.0001). CisR–HeLa/–SiHa was more dependent on pGSK3β, and activation of GSK3 by SMIs (iAkt), treatment with nimbolide, or knockdown of E6/E7 reduced cisplatin resistance and promoted apoptosis. Hence, the activation of GSK3β with nimbolide and iAkt can be exploited for therapeutic interventions of cervical cancer.

hsa_circ_0000129 targets miR‐383‐5p/tropomyosin 3 axis to facilitate ovarian cancer progression

AbstractOvarian cancer is one of the most prevalent malignancies among women. CircRNAs play key roles in the progression of ovarian cancer. This study aimed to investigate the mechanism of action of hsa_circ_0000129 and its effects on ovarian cancer. Expression of hsa_circ_0000129, tropomyosin 3 (TPM3), and miR‐383‐5p in ovarian cancer cell lines and tissue specimens was detected using qRT‐PCR or western blotting. Cell counting kit‐8 (CCK‐8), colony formation, and transwell assays were performed to assess viability, proliferation, and migration of ovarian cancer cells. A xenograft model was used to study tumorigenicity of ovarian cancer cells in vivo. Luciferase and RNA immunoprecipitation assays were performed to determine binding between miR‐383‐5p and hsa_circ_0000129 or TPM3. Upregulation of hsa_circ_0000129 and TPM3 and downregulation of miR‐383‐5p were observed in ovarian cancer. Low hsa_circ_0000129 and TPM3 expression repressed viability, migration, and proliferation of ovarian cancer cells. Inhibition of miR‐383‐5p had a contrary effect. Furthermore, knockdown of hsa_circ_0000129 restricted the tumorigenicity of ovarian cancer cells. Mechanistically, hsa_circ_0000129 has a sponging effect on miR‐383‐5p, which targets TPM3. Hsa_circ_0000129 stimulated development of the malignant ovarian cancer phenotype by sponging miR‐383‐5p and releasing TPM3.

Nimotuzumab and irinotecan synergistically induce ROS‐mediated apoptosis by endoplasmic reticulum stress and mitochondrial‐mediated pathway in cervical cancer

AbstractIrinotecan (CPT‐11), a chemotherapeutic agent used to treat several types of cancer, induces cytotoxic effects on healthy cells. The epidermal growth factor receptor (EGFR) plays a crucial role in various forms of cancer. Nimotuzumab (NmAb), a monoclonal antibody that targets the EGFR, is utilized in some countries to treat malignancies that have an overexpression of EGFR. Yet, there is a lack of literature on the potential anticancer properties of the CPT‐11 and NmAb combination on in vitro human cervical cancer cells. This study investigates the apoptosis mode of the CPT‐11 and NmAb combination on cervical HeLa cancer cells. The Annexin V/propidium iodide staining examination demonstrated that the combination of CPT‐11 and NmAb resulted in a decrease in the number of viable cells and more potent induction of cell apoptosis than the effects of CPT‐11 or NmAb alone in HeLa cells. Furthermore, the combined treatment resulted in elevated levels of reactive oxygen species (ROS) and Ca2+ compared to the treatment with CPT‐11 or NmAb alone. Cells that were pretreated with N‐acetyl‐l‐cysteine, a substance that scavenges ROS, and then treated with CPT‐11, NmAb, or a combination of CPT‐11 and NmAb exhibited higher numbers of viable cells compared to those treated with CPT‐11 or NmAb alone. The combination of CPT‐11 and NmAb resulted in significantly higher caspase‐3, ‐8, and ‐9 activity levels than CPT‐11 or NmAb alone, as measured by flow cytometer assay. The combination of CPT‐11 and NmAb in HeLa cells resulted in elevated endoplasmic reticulum stress‐, mitochondria‐, and caspase‐mediated proteins compared to treatment with CPT‐11 or NmAb alone. According to these observations, NmAb enhances the effectiveness of CPT‐11 in fighting cancer by stimulating cell death in the HeLa cells. Therefore, NmAb has the potential to improve the efficacy of CPT‐11 as a future cervical cancer treatment in humans.

Inhibition of USP21 leads to ovarian carcinoma cell death by suppressing MAPK signaling

AbstractOvarian cancer is the most aggressive and lethal of all gynecologic malignancies. Although the overexpression (OE) of ubiquitin‐specific peptidase 21 (USP21) has been observed in multiple cancers, its expression profile and biological function in ovarian cancer remain unknown. The expression levels of USP21 in ovarian cancer cells and tissues as well as adjacent normal tissues were assessed by qRT‐PCR or Western blot assay. The biological function of USP21 in ovarian cancer cells was assessed by cell growth assay in vitro and a tumor growth model in vivo. Our study revealed that USP21 was markedly elevated in ovarian carcinoma tissues compared with adjacent normal tissues. Downregulation of USP21 attenuated the expression levels of MEK2 and p‐ERK1/2. Depletion of USP21 resulted in suppressed cell growth of ovarian cancers in vitro and inhibited tumor growth in vivo. Conversely, OE of USP21 promoted the cell proliferation of ovarian cancers and conferred resistance to BAY 11‐7082. These findings provide evidences supporting the notion of USP21 as a promising therapeutic target for the treatment of ovarian cancer.

Characterization and validation of fatty acid metabolism‐related genes predicting prognosis, immune infiltration, and drug sensitivity in endometrial cancer

AbstractEndometrial cancer is considered to be the second most common tumor of the female reproductive system, and patients diagnosed with advanced endometrial cancer have a poor prognosis. The influence of fatty acid metabolism in the prognosis of patients with endometrial cancer remains unclear. We constructed a prognostic risk model using transcriptome sequencing data of endometrial cancer and clinical information of patients from The Cancer Genome Atlas (TCGA) database via least absolute shrinkage and selection operator regression analysis. The tumor immune microenvironment was analyzed using the CIBERSORT algorithm, followed by functional analysis and immunotherapy efficacy prediction by gene set variation analysis. The role of model genes in regulating endometrial cancer in vitro was verified by CCK‐8, colony formation, wound healing, and transabdominal invasion assays, and verified in vivo by subcutaneous tumor transplantation in nude mice. A prognostic model containing 14 genes was constructed and validated in 3 cohorts and clinical samples. The results showed differences in the infiltration of immune cells between the high‐risk and low‐risk groups, and that the high‐risk group may respond better to immunotherapy. Experiments in vitro confirmed that knockdown of epoxide hydrolase 2 (EPHX2) and acyl‐CoA oxidase like (ACOXL) had an inhibitory effect on EC cells, as did overexpression of hematopoietic prostaglandin D synthase (HPGDS). The same results were obtained in experiments in vivo. Prognostic models related to fatty acid metabolism can be used for the risk assessment of endometrial cancer patients. Experiments in vitro and in vivo confirmed that the key genes HPGDS, EPHX2, and ACOXL in the prognostic model may affect the development of endometrial cancer.

Spatio‐temporal localization of P21‐activated kinase in endometrial cancer

AbstractEndometrial cancer is the sixth most common gynecologic cancer, and has been reported as a malignancy arising due to the idiopathic effects of certain anticancer agents. Tamoxifen is the drug of choice in ER‐positive breast cancer, and several studies have shown better disease‐free survival in these patients. However, the long‐term usage of tamoxifen has been associated with resistance and risk for endometrial malignancy. A direct mechanistic basis for tamoxifen‐induced endometrial tumorigenesis is still unclear. Hyperactivation of PAK1 in endometrial cancer correlates with poor overall survival. The present study demonstrates that tamoxifen treatment induces nuclear localization of PAK1 in endometrial carcinoma cells. This nuclear transit is mediated through JAK2 phosphorylation of PAK1 and binding of β‐PIX. In addition, a computational approach involving molecular modeling and simulation of phosphorylated and unphosphorylated forms of PAK1 was used to elucidate the dynamics of nuclear localization. Thus, PAK1 phosphorylation by JAK2 is a prerequisite for its nuclear localization and its tumorigenic effects on endometrial cancer cells.

Engineering of Ruthenium‐Based Anticancer Drug RAPTA‐C and Carvacrol Incorporated Hyaluronic Acid‐Altered Liposome Formulation for Ovarian Cells and Its Apoptosis Induction

ABSTRACT Ovarian cancer (OC) is the fifth most common cancer in women, resulting in more deaths than any other female reproductive system disease. This investigation presents hyaluronic acid (HA)‐altered liposome encapsulating 1,3,5‐triaza‐7‐phosphaadamantane compound (RAP) and carvacrol (CAR) (RAP‐HA@Lipo/CAR) for targeted delivery to enhance antitumor and antimetastatic efficacy in OC. The co‐delivered liposomes enhanced anticancer and synergic activity in A2780 OC cells. Further, the apoptotic potential of RAP‐HA@Lipo/CAR was assessed by acridine orange (AO)/propidium iodide (PI) and 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI)/PI staining techniques. The antimetastatic potential of RAP‐HA@Lipo/CAR was evaluated using wound healing and migration assays. Our findings indicated that HA alteration enhanced the cell uptake of the developed RAP‐HA@Lipo/CAR in A2780 cell lines. This study demonstrates a tumor‐targeting RAP‐HA@Lipo/CAR delivery promising to treat OC cells.

Elucidating the anticancerous efficacy of genistein via modulating HPV (E7 and E6) oncogenes expression and apoptotic induction in cervical cancer cells

AbstractIn recent years, genistein has garnered increased interest for its ability to inhibit numerous deregulated targets associated with cancer progression and induction of programmed cell death and antiproliferative activities in human carcinoma cells. Cancer etiology is influenced via multiple disrupted signaling pathways. This study therefore directed toward investigating genistein efficacy in modulating mRNA expression levels of two crucial Human Pappiloma Virus (HPV) (E7 and E6) oncogenes for cancer treatment. Moreover, the inhibitory effects of genistein for HPV (E7 and E6) oncogenes in cervical carcinoma have not yet been reported. Current study investigated inhibitory potential of genistein in HPV (E7 and E6) oncogenes in HeLa cells. These oncogenes are known to deactivate many tumor suppressor proteins (p53 and pRB). Genistein therapy resulted in decreased cell proliferation and increased cell accumulation in the G (G0/G1) phase in HeLa cell lines. In addition, genistein therapy has resulted in the suppression of HPV (E7 and E6) gene expression and simultaneously increasing expression levels of p53 and pRB mRNA levels. As a consequence, there has been an activation of a series of caspases (3, 8, and 9), resulting in their cleavage. Consequently, our data suggests that genistein could be a powerful candidate for treating cervical cancer by targeting two important oncogenes involved in viral development. However, more in vitro research on primary cervical cancer cells is required to validate the clinically relevant efficacy of genistein against cervical cancer.

RBPMS‐AS1 sponges miR‐19a‐3p to restrain cervical cancer cells via enhancing PLCL1‐mediated pyroptosis

AbstractCervical cancer (CC) poses a threat to human health. Enhancing pyroptosis can prevent the proliferation and epithelial–mesenchymal transition (EMT) of tumor cells. This study aims to reveal the candidates that modulate pyroptosis in CC. Accordingly, the common microRNAs (miRNAs/miRs) that were sponged by RBPMS antisense RNA 1 (RBPMS‐AS1) and could target Phospholipase C–Like 1 (PLCL1) were intersected. The expression of PBPMS‐AS1/miR‐19a‐3p (candidate miRNA)/PLCL1 was predicted in cervical squamous cell carcinoma (CESC), by which the expression location of RBPMS‐AS1 and the binding between RBPMS‐AS1/PLCL1 and miR‐19a‐3p were analyzed. The targeting relationship between RBPMS‐AS1/PLCL1 and miR‐19a‐3p was confirmed by dual‐luciferase reporter assay. After the transfection, cell counting kit‐8 assay, colony formation assay, quantitative reverse transcription PCR, and Western blot were implemented for cell viability and proliferation analysis as well as gene and protein expression quantification analysis. Based on the results, RBPMS‐AS1 and PLCL1 were lowly expressed, yet miR‐19a‐3p was highly expressed in CESC. RBPMS‐AS1 overexpression diminished the proliferation and expressions of N‐cadherin, vimentin, and miR‐19a‐3p, yet enhanced those of E‐cadherin, PLCL1, and pyroptosis‐relevant proteins (inteleukin‐1β, caspase‐1, and gasdermin D N‐terminal). However, the above RBPMS‐AS1 overexpression–induced effects were counteracted in the presence of miR‐19a‐3p. There also existed a targeting relationship and negative interplay between PLCL1 and miR‐19a‐3p. In short, RBPMS‐AS1 sponges miR‐19a‐3p and represses the growth and EMT of CC cells via enhancing PLCL1‐mediated pyroptosis.

HPV18 E6/E7 activates Ca2+ influx to promote the malignant progression of cervical cancer by inhibiting Ca2+ binding protein 1 expression

AbstractMounting studies have shown that the oncoproteins E6 and E7 encoded by the human papillomavirus (HPV) genome are essential in HPV‐induced cervical cancer (CC). Ca2+ binding protein 1 (CABP1), a downstream target of HPV18‐positive HeLa cells that interferes with E6/E7 expression, was identified through screening the GEO Database (GSE6926). It was confirmed to be down‐regulated in CC through TCGA prediction and in vitro detection. Subsequent in vitro experiments revealed that knocking down E6/E7 inhibited cell proliferation, migration, and invasion, whereas knocking down CABP1 promoted these processes. Simultaneously knocking down CABP1 reversed these effects. Additionally, the results were validated in vivo. Previous studies have indicated that CABP1 can regulate Ca2+ channels, influencing Ca2+ influx and tumor progression. In this study, it was observed that knocking down CABP1 enhanced Ca2+ inflow, as demonstrated by flow cytometry and confocal microscopy. Knocking down E6/E7 inhibited these processes, whereas simultaneously knocking down E6/E7 and CABP1 restored the inhibitory effect of knocking down E6/E7 on Ca2+ inflow. To further elucidate that E6/E7 promotes CC progression by inhibiting CABP1 expression and activating Ca2+ influx, BAPTA/AM treatment was administered during CABP1 knockdown. It was discovered that Ca2+ chelation could reverse the effect of CABP1 knockdown on CC cells. In conclusion, our results offer a novel target for the diagnosis and treatment of HPV‐induced CC.

The impact of oleuropein on miRNAs regulating cell death signaling pathway in human cervical cancer cells

AbstractCervical cancer is known as the second most pervasive malignancy in women across the globe. The role played by microRNAs (miRNAs) in the initiation, progression, and metastasis of this cancer has received specific attention. The use of natural compounds leading cancer cells toward apoptosis is a feasible strategy for cancer therapy. Oleuropein, an olive‐extracted phenolic substance, displays anticancer properties. Here, it was attempted to assess the role played by oleuropein in cell viability in cervical cancer and changes in the expression of some miRNAs associated with cervical cancer as well as some of their possible target genes selected using bioinformatics analysis. For this purpose, HeLa cell line was exposed to several oleuropein concentrations for 48 and 72 h. After that, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay and flow cytometry were employed to assess cell viability and apoptosis, respectively. In addition, to conduct bioinformatics analysis, Cytoscape computer program was used based on STRING database. Furthermore, to examine the role played by oleuropein in the expression of miRNAs of interest as well as their potential target genes, real‐time PCR was employed. The findings indicated that oleuropein reduced cell viability through inducing apoptosis. As a result of treatment with oleuropein, miR‐34a, miR‐125b, and miR‐29a showed increased expression levels, whereas miR‐181b, miR‐221, and miR‐16 showed decreased expression levels. Furthermore, oleuropein reduced the expression of the anti‐apoptotic genes Bcl‐2 and Mcl1, whereas it elevated the expression of the pro‐apoptotic Bid, Fas, and TNFRSF10B genes and the p53 tumor suppressor. Our results indicate that the apoptosis induction is a mechanism of action of oleuropein in HeLa cells. Because of its effect on the reflation of the expression of genes and miRNAs effective in the pathogenesis of cervical cancer, oleuropein shows potential as an effective research tool for developing new natural drugs for treating cervical cancer.