Cell Biochemistry and Function

E7HPV16 Oncogene and 17beta‐Estradiol Stress, Promotes Oncogenic microRNA Expression Patterns, Cell Proliferation and Cervical Intraepithelial Neoplasia 1

ABSTRACTCervical cancer (CC) is the second cause of death by a neoplasia in woman in Mexico. Among the factors that contribute to its development are prolonged infection by a high‐risk HPV type and the use of estrogens. It is well known that diagnosis at early stages is extremely important since, in most cases, progression towards carcinogenesis could be prevented, hence the importance of finding candidates that serve as early biomarkers. Several studies have shown that the expression level of the tumor suppressor miR‐218 is diminished in CC while oncomiR miR‐21 is overexpressed. On the other hand, it has been reported that the Potassium calcium‐activated channel subfamily M alpha 1 (Kcnma1) oncogene, a known target gene of miR‐218, is overexpressed in CC. However, there are few studies on the expression of this oncogene in Cervical Intraepithelial Neoplasia 1 (CIN 1). In this study, the analysis of the K14E7HPV16 carcinogenesis model in young mice (1.5‐month‐old), showed that a single‐dose of 17β‐estradiol (E2) increased both the cell proliferation and the Bcl‐2 oncogene expression, as well as promoted the development of CIN 1. Interestingly, the hormonal stress and the E7 expression, favor the physiological response of the organism in transgenic young mice by decreasing the expression levels of the tumor suppressor miR‐218 and increasing the expression of the Kcnma1 and Bcl‐2 mRNA oncogenes in both, cervical tissue and serum. This work demonstrates the significance of a single E2 stimulation and the expression of the HPV E7 oncoprotein in the early stage of cervical carcinogenesis. In addition, we provide strong evidence about Kcnma1 oncogene as a target gene of miR‐218 and that both could be used as early circulating biomarkers of CC.

Ovarian cancer derived PKR1 positive exosomes promote angiogenesis by promoting migration and tube formation in vitro

Cancer cell derived exosomes play important roles in cancer progression and modulation of the tumour microenvironment. This study aims to investigate the role of prokineticin receptor 1 (PKR1) positive exosomes on angiogenesis. In the present study, PKR1 expression in tumour samples from ovarian cancer patients were examined firstly. Then, two ovarian cancer cell lines, namely A2780 and HO‐8910 cells, were used to isolate and obtain the PKR1 positive exosomes from the serum free medium. The function analysis of PKR1 positive exosomes on angiogenesis was conducted by cell proliferation and migration assay, tube formation analysis, and tumour volume assay. The results showed that PKR1 expression was down regulated in tumour samples of ovarian cancer patients compared with adjacent normal tissues. The intracellular expression of PKR1 could be detected in A2780 and HO‐8910 cells. And, the isolated exosomes from the serum free medium were confirmed by transmission electron microscopic and NTA analysis, as well as the co‐presence of PKR1 with exosome marker CD63. The function analysis of PKR1 positive exosomes on angiogenesis demonstrated the uptake of PKR1 positive exosomes by human umbilical vein endothelial cells through immunofluorescence staining. The angiogenesis assays in vitro indicated that PKR1 positive exosomes promoted migration and tube formation of HUVECs but not proliferation. The endogenous PKR1 was also verified to help to enhance migration and promote tube formation of vascular endothelial cells, which might involved in the phosphorylation of STAT3. Additionally, The tumour volume from exosomes treated A2780 tumour‐bearing mice was significantly increased compared with the control group, accompanied with the induced PKR1 expression and phosphorylation of STAT3 level.Significance of the StudyThis study proved the important role of PKR1 positive exosomes released from ovarian cancer cells on promoting angiogenesis. The data indicated that PKR1 derived from ovarian cancer cells could act as an important tumour associated antigen and biomolecular factor for cellular communication in tumour microenvironment.

Copy number and expression of CEP89, a protein required for ciliogenesis, are increased and predict poor prognosis in patients with ovarian cancer

AbstractCEP89 (centrosomal protein 89) is required for ciliogenesis and mitochondrial metabolism, but its role in cancer has yet to be clarified. We report that CEP89 is overexpressed in ovarian cancer (OC) compared to normal ovaries. Likewise, its expression is higher in malignant ovarian tumors than in borderline ovarian tumors with low malignant potential. More than a quarter of patients with OC have copy number gains in the CEP89 gene, and patients with high expression have more than a year shorter overall survival compared to those with low expression. Moreover, we found that CEP89 can be considered as a prognostic marker for poor overall survival in patients with OC, after adjusting for tumor stage and residual tumor. Nine out of the top 10 protein interactors of CEP89 have the highest percentage of total copy number variation (CNV) events in OC among all other cancer types. Furthermore, CEP89 messenger RNA (mRNA) levels are higher in OC patients with disease recurrence compared to those with no recurrence. We also analyzed CEP89 levels in OC cell lines in terms of CNV, mRNA, and protein levels; and observed that the FUOV‐1 cell line has the highest levels among cell lines that originated from primary sites. Our study suggests that CEP89 may be a valuable prognostic predictor for the overall survival of patients with OC, and it could also be a novel therapeutic target in this malignancy.

LINC00662 contributes to the progression and the radioresistance of cervical cancer by regulating miR‐497‐5p and CDC25A

It is reported that long intergenic non‐coding RNA 00662 (LINC00662) plays an oncogenic role in tumours. However, the mechanism of LINC00662 in regulating the progression and radiosensitivity of cervical cancer (CC) is not clear. In this study, quantitative real‐time polymerase chain reaction (qRT‐PCR) was adopted to detect LINC00662 and miR‐497‐5p expressions in CC tissues and cells. The expression of cell division cycle 25 A (CDC25A) in CC cells was examined by Western blot. CC cell proliferation was determined by cell counting kit‐8 (CCK‐8) and BrdU assays. The survival rate of CC cells was evaluated by colony formation assay under different doses of X‐ray irradiation. CC cell migration and invasion were probed by Transwell assay. Besides, the interactions between miR‐497‐5p and LINC00662, and miR‐497‐5p and the 3′UTR of CDC25A were verified by dual‐luciferase reporter assay, RIP assay, and RNA pull‐down experiments. We demonstrated that, LINC00662 expression was remarkably raised in CC tissues and cell lines. LINC00662 overexpression promoted proliferation, migration, invasion and radioresistance of CC cells, and LINC00662 knockdown inhibited the above malignant phenotypes of CC cells. In terms of mechanism, LINC00662 facilitated CC progression and radioresistance by adsorbing miR‐497‐5p and indirectly up‐regulating CDC25A expression. In a word, the LINC00662/miR‐497‐5p/CDC25A axis boosts proliferation and metastasis of CC cells and enhances the radioresistance of cancer cells.Significance of the studyCC poses a threat to the health of women all over the world. In this study, we demonstrated for the first time that LINC00662 expression was remarkably raised in CC tissues and cells. Cellular experiments confirmed that LINC00662 facilitated cell proliferation, migration, invasion and radiation resistance through the miR‐497‐5p/CDC25A axis, which might be a promising target for CC treatments.

A three‐dimensional microenvironment alters CD73 expression in cervical cancer

Stem‐like cells (CSCs) have a tumour‐initiating capacity and play critical role in tumour metastasis, relapse and resistance to therapy. The ectoenzyme CD73, encoded by the NT5E gene, which catalyses the hydrolysis of AMP into adenosine, has been associated to an immunosuppressive tumour microenvironment, tumour cell adhesion and migration. Therefore, we investigated the expression and activity of CD73 in sphere‐forming cells from cervical cancer in comparison to monolayer cells in vitro. In addition, in silico analysis was performed to determine the expression of CD73 and other members of purinergic signalling in CSC‐like population derived from different tumour types in comparison to monolayer cells. CD73 protein expression levels and functionality in SiHa cells were analysed by flow cytometry and enzymatic assay, respectively. In silico investigation was performed through the analysis of seven datasets from different tumour types using GEO database. In vitro analysis showed a decreased CD73 protein expression and enzymatic activity in cervical spheres, when compared to monolayers. In addition, when sphere‐derived cells are re‐plated as monolayer culture, the CD73 expression and activity are restored. Supporting the in vitro results, in silico analysis showed that three‐dimensional spheres derived from cervical, thyroid and breast cancer presented decreased expression of CD73, when compared to their adherent counterparts. The decreased expression of CD73 in sphere‐derived cells or CSC‐enriched population reinforce its important role in cell adhesion, tumour spreading ability and metastasis, suggesting CD73 as potential target to be further investigated in cervical cancer.

DGUOK‐AS1 promotes cell proliferation in cervical cancer via acting as a ceRNA of miR‐653‐5p

Cervical cancer (CC) holds the second highest incidence and is the fourth dominating cause of cancer‐induced death in women. It has been widely accepted that long noncoding RNAs (lncRNAs) are implicated in pathological and physiological activities of CC. However, the research of lncRNAs is still in the initial stage. The biological function of lncRNA deoxyguanosine kinase antisense RNA 1 (DGUOK‐AS1) in human cancers has not been reported yet. We found that DGUOK‐AS1 was aberrantly upregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tissues through TCGA database. Real‐time quantitative polymerase chain reaction (RT‐qPCR) also verified the high expression of DGUOK‐AS1 in CC cell lines. Loss‐of‐function assays indicated that DGUOK‐AS1 silence repressed CC cell growth. In addition, dual‐luciferase reporter and RNA immunoprecipitation (RIP) experiments validated the binding relation between miR‐653‐5p and DGUOK‐AS1 or EMSY. Results of the rescue assays elucidated that EMSY overexpression or miR‐653‐5p downregulation reversed the suppressive function of DGUOK‐AS1 knockdown on cell growth and DNA repair in CC. To sum up, this research highlighted that DGUOK‐AS1 could promote CC cell proliferation via serving as a ceRNA of miR‐653‐5p to release EMSY, which might inspire us to discover novel strategies for CC treatment.Significance of the studyDGUOK‐AS1 knockdown hinders proliferation of CC cells. DGUOK‐AS1 sequesters miR‐653‐5p to elevate EMSY in CC. EMSY is required for DGUOK‐AS1 to induce cell proliferation and repress DNA damage in CC.

TSPAN31 suppresses cell proliferation in human cervical cancer through down‐regulation of its antisense pairing with CDK4

Natural antisense transcripts (NAT) are prevalent phenomena in the mammalian genome and play significant regulatory roles in gene expression. While new insights into NAT continue to be revealed, their exact function and their underlying mechanisms in human cancer remain largely unclear. We identified a NAT of CDK4, referred to TSPAN31, which inhibits CDK4 mRNA and protein expression in human cervical cancer by targeting the 3′‐untranslated region (3′‐UTR) of the CDK4 mRNA. Furthermore, silencing the expression of the TSPAN31 mRNA rescued the TSPAN31 3′‐UTR‐ or the TSPAN31 full‐length‐induced decrease in CDK4 expression. Noteworthy, we discovered that TSPAN31, as a member of the tetraspanin family, suppressed cell proliferation by down‐regulating its antisense pairing with CDK4 and decreasing retinoblastoma protein phosphorylation in human cervical cancer. Therefore, the results of the present study suggest that TSPAN31 may serve as a potential molecular target for the development of novel anti‐cancer agents.Significance of the studyNatural antisense transcripts are widely found in the genome and play an important role in the growth and development of cells. TSPAN31 is natural antisense transcript, and CDK4 is an important gene in the regulation of the cell cycle. Therefore, TSPAN31 and CDK4 have great significance in the study of tumour therapeutic targets.

Eupatilin regulates proliferation and cell cycle of cervical cancer by regulating hedgehog signalling pathway

Eupatilin (5,7‐dihydroxy‐3′,4′,6‐trimethoxyflavone) is a natural active substance found in génépi group plants, and its pharmacological activities has been proven to be useful in the treatment of various cancers. However, whether eupatilin demonstrates anti‐cancer activity in cervical cancer is still under evaluation. To clarify this, cancer cell lines and nude mouse model were used in this study. The results indicated that eupatilin could inhibit the occurrence of cervical cancer both in vivo and in vitro. Cervical cancer cell lines (C4‐1, HeLa, Caski, and Siha) and Ect1/E6E7 cells were incubated with eupatilin (40μM) for 48 hours. Compared with the control group, the viability of cervical cancer cells decreased significantly, while the apoptotic cells increased significantly. Cell cycle analysis showed that eupatilin treatment of HeLa and Caski cells reduced the proliferation index. Eupatilin at 40 mg/kg also inhibited tumour growth in tumour‐bearing mice. Interestingly, weakened hedgehog signalling was observed in cervical cancer cells and tumours from tumour‐bearing mice after eupatilin treatment. Our results reveal the inhibitory effect of eupatilin on cervical cancer and shed new light on the molecular mechanism of its therapeutic effect.Significance of the studyEupatilin inhibited proliferation via promoting apoptosis and cell cycle arrest in HeLa and Caski cervical cancer cell lines. In addition, nude mouse tumourigenicity assay proved that eupatilin can suppress tumour growth in vivo. Dramatically, these activities might be involved in hedgehog signal pathway.

Actin Gamma Smooth Muscle 2 Promotes Epithelial Ovarian Cancer Cell Proliferation via the AKT1/NF‐κB Signaling Pathway

ABSTRACTEpithelial ovarian cancer (EOC) is associated with high mortality rates worldwide and is characterized as the most lethal gynecological cancer. The study aimed to investigate the functional role and underlying molecular mechanism of actin gamma smooth muscle 2 (ACTG2) in the progression of EOC. Data mining from The Cancer Genome Atlas (TCGA) databases showed the expression of ACTG2 was significantly upregulated in EOC and negatively associated with longer overall survival and better prognosis of patients. By using of gain‐of‐function and loss‐of‐function experiments in vitro and in vivo, we found that ACTG2 promoted EOC cell proliferation and suppressed cell apoptosis. Mechanistic study revealed that ACTG2 regulates EOC cell proliferation by activating the AKT serine/threonine kinase 1 (AKT1)/nuclear factor‐κB (NF‐κB) signaling pathway. Importantly, p65 plays a crucial role in this newly identified regulatory mechanism. Our findings demonstrate that ACTG2 may play an oncogenic role in EOC, suggesting its potential as a therapeutic target.

Nucleic acid‐based vaccine for ovarian cancer cells; bench to bedside

AbstractOvarian cancer continues to be a difficult medical issue that affects millions of individuals worldwide. Important platforms for cancer immunotherapy include checkpoint inhibitors, chimeric antigen receptor T cells, bispecific antibodies, cancer vaccines, and other cell‐based treatments. To avoid numerous infectious illnesses, conventional vaccinations based on synthetic peptides, recombinant subunit vaccines, and live attenuated and inactivated pathogens are frequently utilized. Vaccine manufacturing processes, however, are not entirely safe and carry a significant danger of contaminating living microorganisms. As a result, the creation of substitute vaccinations is required for both viral and noninfectious illnesses, including cancer. Recently, there has been testing of nucleic acid vaccines, or NAVs, as a cancer therapeutic. Tumor antigens (TAs) are genetically encoded by DNA and mRNA vaccines, which the host uses to trigger immune responses against ovarian cancer cells that exhibit the TAs. Despite being straightforward, safe, and easy to produce, NAVs are not currently thought to be an ideal replacement for peptide vaccines. Some obstacles to this strategy include selecting the appropriate therapeutic agents (TAs), inadequate immunogenicity, and the immunosuppressive characteristic of ovarian cancer. We focus on strategies that have been employed to increase NAVs' effectiveness in the fight against ovarian cancer in this review.

Knockdown of Trop2 inhibits proliferation and migration and induces apoptosis of endometrial cancer cells via AKT/β‐catenin pathway

Endometrial cancer (EC) is the most common gynaecologic malignancy in western countries and has been reported to account for about 7% of female malignant tumours and 20% to 30% of female genital system malignant tumours. Accumulating evidence showed the expression of human trophoblast cell surface antigen 2 (Trop2) was abnormal in many cancers; however, the expression and role of Trop2 in EC are not clear. The Trop‐2 protein expression was detected by western blot in EC cells. Cell proliferation, apoptosis, and migration were measured by CCK‐8, flow cytometry, and Transwell assay, respectively. The epithelial mesenchymal transition (EMT) and AKT/β‐catenin signalling pathway–related proteins in EC cell lines were detected by western blot assay following Trop2 gene silencing. The present study revealed that the Trop2 protein was highly expressed in EC cell lines compared with human endometrial epithelial cells. The Trop2 mRNA and protein were obviously decreased following transfection with Trop2‐siRNA sequence in KLE and Ishikawa cells. Meanwhile, Trop2 gene silencing in KLE and Ishikawa cells strongly inhibited cell proliferation and migration and increased cell apoptosis. Investigation into the molecular mechanism indicated that the Trop2 gene silencing suppressed EMT and AKT/β‐catenin signalling pathway activation.Significance of the studyThese findings suggested that Trop2 silencing inhibited EC cell proliferation and migration and promoted cell apoptosis. The mechanism might be related to the inhibition of the AKT/β‐catenin signalling pathway in EC cells. Therefore, Trop2 may be a potential therapeutic target for the treatment of EC.

Autophagy‐related gene PXN as a prognostic marker: Promotion of ovarian cancer progression by suppressing the p110β/Vps34/Beclin1 pathway

AbstractAmong gynecological malignancies, ovarian cancer has the highest mortality rate and has sparked widespread interest in studying the mechanisms underlying ovarian cancer development. Based on TCGA and GEO databases, we investigated the highly expressed autophagy‐related genes that determine patient prognosis using limma differential expression and Kaplan–Meier survival analyses. The biological processes associated with these genes were also predicted using GO/KEGG functional enrichment analysis. CCK‐8, cell scratch, and transwell assays were used to investigate the effects of PXN on the proliferation, migration, and invasion abilities of ovarian cancer cells. Transmission electron microscopy was used to observe the autophagosomes. The expression of autophagy proteins and the PI3K/Akt/mTOR and p110β/Vps34/Beclin1 pathway proteins in ovarian cancer cells was detected using western blot; autophagy protein expression was further detected and localized using cellular immunofluorescence. A total of 724 autophagy‐related genes were found to be overexpressed in ovarian ‐cancer tissues, with high expression of PEX3, PXN, and RB1 associated with poor prognosis in patients (p < .05). PXN activates and regulates signaling pathways related to cellular autophagy, ubiquitination, lysosomes, PI3K‐Akt, and mTOR. Autophagosomes were observed in all cell groups. The increase in PXN gene expression promoted the proliferation, migration, and invasion of ovarian cancer cells, increased the expression of SQSTM1/p62 protein, decreased LC3II/LC3Ⅰ, inhibited the phosphorylation of Akt and mTOR proteins, and suppressed the expression of PI3K(p110β) and Beclin1 proteins. The decrease in PXN expression also confirmed these changes. Thus, PXN is highly expressed during ovarian cancer and is associated with poor patient prognosis. It may promote ovarian cancer cell proliferation, migration, and invasion by inhibiting cellular autophagy via suppression of the p110β/Vps34/Beclin1 pathway.

Expression of Concern: LncRNA TUG1 facilitates proliferation, invasion and stemness of ovarian cancer cell via miR‐186‐5p/ZEB1 axis

LncRNA TUG1 has been rarely studied in ovarian cancer (OC), our objective was to explore the role of TUG1 in the regulation of malignant phenotypes of OC. Vectors of sh‐TUG1, miR‐186‐5p and pcDNA‐ZEB1 were, respectively, constructed and used to infect OC cells. MTT and transwell assays were applied for representing cell proliferation and invasion, respectively. Sphere formation experiment was used to detect the stemness of OC cells. Western blotting and qRT‐PCR were employed for detecting the expression of multiple biomarkers on protein and RNA levels, respectively. The luciferase assay was performed to reveal the interactions between miR‐186‐5p and TUG1 or ZEB1. The silencing of TUG1 and upregulation of miR‐186‐5p both suppressed the cell proliferation, invasion and cancer stem cell (CSC) properties. Additionally, luciferase assay verified that miR‐186‐5p directly binds TUG1 and ZEB1. Moreover, overexpression of ZEB1 rescued the impact on the proliferation, invasion and stemness of TUG1 silencing in OC. TUG1 sponges miR‐186‐5p to release ZEB1 and promotes the proliferation, invasion and stemness of OC cells, suggesting that TUG1 could be a potential therapeutic target for OC therapy.Significance of the studyLncRNA TUG1 could promote proliferation, invasion and stemness of ovarian cancer cells. Our study first discovered that TUG1 play a tumourigenic role in ovarian cancer by regulating stemness of cancer cells. Mechanism research exhibited the regulation role of TUG1 in ovarian cancer cells was miR‐186‐5p/ZEB1 axis depended. These results provided a new perspective to understand the pathogenesis and development of ovarian cancer; it will offer new evidence for better diagnosis and treatment therapy of ovarian cancer.

PDLIM2 can inactivate the TGF‐β/Smad pathway to inhibit the malignant behavior of ovarian cancer cells

AbstractPDZ‐LIM domain‐containing Protein 2 (PDLIM2) has been reported to be downregulated in ovarian cancer. However, its exact function and mechanism in regulating ovarian cancer progression have not been elucidated. This work researched the exert effect and mechanism of PDLIM2 on ovarian cancer progression. Briefly, PDLIM2 expression in clinical tissues of ovarian cancer patients and cells was investigated by qRT‐PCR and Western blot. The function of PDLIM2 on the proliferation, colony formation, migration and invasion of ovarian cancer cells was explored via cell counting kit‐8, colony formation and Transwell assays. To verify whether PDLIM2 regulates ovarian cancer progression via regulating the transforming growth factor‐β (TGF‐β)/Smad pathway, exogenous TGF‐β (10 ng/mL) treatment was performed on the PDLIM2‐overexpressed ovarian cancer cells. PDLIM2 effect on the in vivo growth of ovarian cancer cells was researched by establishing a xenograft tumor model. Immunohistochemistry and Western blot were performed to protein expression in cells and tissues. As a result, PDLIM2 was low‐expressed in ovarian cancer tissues/cells. PDLIM2 upregulation attenuated the proliferation, colony formation, migration, invasion and epithelial‐mesenchymal transition (EMT) of ovarian cancer cells, and inactivated the TGF‐β/Smad pathway. The opposite results were found in the PDLIM2‐silenced ovarian cancer cells. Exogenous TGF‐β treatment abrogated the inhibition of PDLIM2 on the malignant behavior of ovarian cancer cells. PDLIM2 upregulation attenuated the in vivo growth and EMT of ovarian cancer cells. Thus, PDLIM2 attenuates the proliferation, migration, invasion and EMT of ovarian cancer cells via inactivating the TGF‐β/Smad pathway. PDLIM2 may be a usefully target for ovarian cancer treatment.

Adenosine increases PD‐L1 expression in mesenchymal stromal cells derived from cervical cancer through its interaction with A2AR/A2BR and the production of TGF‐β1

AbstractMesenchymal stromal cells (MSCs) together with malignant cells present in the tumor microenvironment (TME), participate in the suppression of the antitumor immune response through the production of immunosuppressive factors, such as transforming growth factor beta 1 (TGF‐β1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF‐β1 by interacting with A2AR/A2BR. In the present study, we provide evidence that Ado induces the production of TGF‐β1 in MSCs derived from CeCa tumors (CeCa‐MSCs) by interacting with both receptors and that TGF‐β1 acts in an autocrine manner to induce the expression of programmed death ligand 1 (PD‐L1) in CeCa‐MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T lymphocytes. The addition of the antagonists ZM241385 and MRS1754, specific for A2AR and A2BR, respectively, or SB‐505124, a selective TGF‐β1 receptor inhibitor, in CeCa‐MSC cultures significantly inhibited the expression of PD‐L1. Compared with CeCa‐MSCs, MSCs derived from normal cervical tissue (NCx‐MSCs), used as a control and induced with Ado to express PD‐L1, showed a lower response to TGF‐β1 to increase PD‐L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF‐β1, and the induction of PD‐L1 in CeCa‐MSCs to suppress the antitumor response of CD8+ T lymphocytes. The findings of this study suggest that this pathway may have clinical importance as a therapeutic target.

Regulation of HeLa cell proliferation and apoptosis by bovine lactoferrin

AbstractCervical cancer is one of the foremost common cancers in women. Lactoferrin (LF) has many biological functions, such as antitumor. This study aimed to explore the regulatory effect of bovine lactoferrin (bLF) on the proliferation and apoptosis of cervical cancer HeLa cells and to clarify the potential mechanism of action of bLF against HeLa cells. This study used CCK‐8, Trypan blue staining, and colony formation assays to verify the effect of bLF on HeLa cell proliferation. Hoechst 33258 fluorescence staining, AO/EB staining, and western blotting were used to determine the effects of bLF on apoptosis and autophagy in HeLa cells. We discovered that bLF significantly reduced the proliferation of HeLa cells in a dose‐ and time‐dependent manner compared to the control group. Furthermore, bLF primarily induced apoptosis in HeLa cells by increasing the expression of the proapoptotic proteins p53, Bax, and Cleaved‐caspase‐3 and downregulating the expression of the antiapoptotic protein Bcl‐2. In addition, the present study also showed that bLF treatment significantly activated autophagy‐related proteins LC3B‐II and Beclin I and down regulated the autophagosome transporter protein p62, indicating that bLF treatment can induce autophagy in HeLa cells. After pretreatment with the autophagy inhibitor, 3‐MA, which markedly found that autophagy inhibition by 3‐MA reversed bLF‐induced apoptosis, indicating that bLF can induce apoptosis by activating intracellular autophagy in HeLa cells. In the present study, our results support the theory of bLF significantly inhibited the proliferation of Hela cells by promoting apoptosis and reinforcing autophagy. The study will play an important role in therapying cervical cancer.

Screening of Cu4O3 NPs efficacy and its anticancer potential against cervical cancer

AbstractCu4O3 is the least explored copper oxide, and its nanoformulation is anticipated to have important therapeutic potential especially against cancer. The current study aimed to biosynthesize Cu4O3 nanoparticles (NPs) using an aqueous extract of pumpkin seeds and evaluate its antiproliferative efficacy against cervical cells after screening on different cancer cell lines. The obtained NPs were characterized by different spectroscopic analyses, such as UV‐vis, thermogravimetric, energy dispersive X‐ray, and Fourier‐transform infrared spectroscopy (FTIR). In addition, high‐resolution transmission electron microscopes (HR‐TEM) were used to observe the morphology of the biosynthesized NPs. The UV‐vis spectra showed a peak at around 332 nm, confirming the formation of Cu4O3 NPs. Moreover, FTIR and TAG analyses identified the presence of various bioactive phytoconstituents that might have worked as capping and stabilization agents and comparative stable NPs at very high temperatures, respectively. The HR‐TEM data showed the spherical shape of Cu4O3 NPs in the range of 100 nm. The Cu4O3 NPs was screened on three different cancer cell lines viz., Hela, MDA‐MB‐231, and HCT‐116 using cytotoxicity (MTT) reduction assay. In addition, Vero was taken as a normal epithelial (control) cell. The high responsive cell line in terms of least IC50 was further assessed for its anticancer potential using a battery of biological tests, including morphological alterations, induction of apoptosis/ROS generation, regulation of mitochondrial membrane potential (MMP), and suppression of cell adhesion/migration. Vero cells (control) showed a slight decline in % cell viability even at the highest tested Cu4O3 NPs concentration. However, all the studied cancer cells viz., MDA‐MB‐231, HCT 116, and HeLa cells showed a dose‐dependent decline in cell viability after the treatment with Cu4O3 NPs with a calculated IC50 value of 10, 11, and 7.2 µg/mL, respectively. Based on the above data, Hela cells were chosen for further studies, that showed induction of apoptosis from 3.5 to 9‐folds by three different staining techniques acridine orange/ethidium bromide (AO/EB), 4′,6‐diamidino‐2‐phenylindole (DAPI), and propidium iodide (PI). The enhanced production of reactive oxygen species (>3.5‐fold), modulation in MMP, and suppression of cell adhesion/migration were observed in the cells treated with Cu4O3 NPs. The current study obtained the significant antiproliferative potential of Cu4O3 NPs against the cervical cancer cell line, which needs to be confirmed further in a suitable in vivo model. Based on our results, we also recommend the green‐based, eco‐friendly, and cost‐effective alternative method for synthesizing novel nanoformulation.

A broad analysis in clinical and in vitro models on regulator of G‐protein signalling 10 regulation that is associated with ovarian cancer progression and chemoresistance

Ovarian cancer is one of the deadliest types of gynaecological cancers and more than half of the patients die within 5 years after diagnosis. Recurrence in advanced staged patients after chemotherapy is associated with increased chemoresistance, which results in poor prognosis. Regulator of G‐protein signalling 10 (RGS10) negatively regulates cell proliferation, migration and survival by attenuating G‐protein coupled‐receptors mediated signalling pathways. Recent studies have shown that loss of RGS10 expression is significantly associated with proliferation and cisplatin resistance in ovarian cancer cells.Significance of the studyIn this study, we analysed differential RGS10 expression levels using public microarray datasets from clinical and in vitro ovarian cancer samples. We validated that cancer progression and chemotherapy exposure change RGS10 expression. We enriched our study to evaluate the relationship between chemoresistance and differential RGS10 expression against ovarian cancer potential chemotherapeutic agent, palbociclib. Results showed that palbociclib treatment reduced cell viability, despite significantly decreased RGS10 expression in chemoresistant cells. Overall, the results confirmed that cancer progression and chemoresistance are significantly associated with the down‐regulation of RGS10 while some chemotherapeutics seem to be beneficial in decreasing chemoresistance in ovarian cancer.

LINC‐PINT suppresses tumour cell proliferation, migration and invasion through targeting miR‐374a‐5p in ovarian cancer

LncRNA LINC‐PINT acts as an important regulator in the development of many cancers. The current study aimed to explore the role of LINC‐PINT in the progression of ovarian cancer (OC). LINC‐PINT expression level in different FIGO stages of OC and its adjacent tissues, normal HOSE and OC cell lines (A2780, SKOV3, OVCAR3 and HO‐8910) was determined by qRT‐PCR. Survival analysis on LINC‐PINT and OC patients was conducted by Kaplan‐Meier. CCK‐8, flow cytometry, wound‐healing, Transwell assays and western blot were performed to detect the effects of LINC‐PINT on proliferation, apoptosis, migration, invasion and EMT process in OC cells. Target gene of LINC‐PINT was predicted by Starbase and verified by dual‐luciferase reporter assay. The expression of miR‐374a‐5p in normal and OC tissues, LINC‐PINT‐ or siLINC‐PINT‐modified OC cells was determined. Moreover, rescue assay was carried out to confirm whether LINC‐PINT contributes to the development of OC cells through targeting miR‐374a‐5p. Low expression of LINC‐PINT was observed in OC tissues and cells, noticeably, LINC‐PINT expression was even lower in OC tissues with higher FIGO stage. Increased LINC‐PINT expression significantly inhibited cell proliferation, promoted apoptosis and suppressed migration, invasion and EMT process, while silencing of LINC‐PINT caused the opposite results. Moreover, LINC‐PINT sponged miR‐374a‐5p and overexpressed miR‐374a‐5p attenuated the effect of up‐regulated LINC‐PINT on cell viability, migration, invasion and apoptosis. LINC‐PINT acts as a tumour suppressor, as it could inhibit cell proliferation, migration, invasion and EMT process, and promote cell apoptosis through down‐regulating miR‐374a‐5p.Significance of the studyOvarian cancer (OC), which is a frequently diagnosed tumour in female reproductive organs, has a high incidence rate behind cervical cancer and endometrial cancer. LncRNA LINC‐PINT acts as an important regulator in the development of many cancers. The current study aimed to explore the role of LINC‐PINT in the progression of ovarian cancer (OC) and found that LINC‐PINT inhibited cell proliferation, migration invasion and EMT process of OC cell via regulating miR‐374a‐5p; it might be a potential target for OC treatment.

DMBT1 suppresses cell proliferation, migration and invasion in ovarian cancer and enhances sensitivity to cisplatin through galectin‐3/PI3k/Akt pathway

Ovarian cancer (OC) is one of the most common gynaecologic malignancies. Deleted in malignant brain tumors 1 (DMBT1) was considered as a tumour suppressor in multiple cancers, but there have been no systemic profiling studies of DMBT1 in OC until now. The aim of this study is to explore the role and the potential mechanism of DMBT1 in OC. mRNA levels and protein expressions of corresponding genes were detected by quantitative real‐time polymerase chain reaction and western blot. Cell proliferation was detected by CCK‐8 assay and cell colony formation. Cell migration and invasion were detected by wound healing and transwell assay. The combination between DMBT1 and galectin‐3 was demonstrated by immunoprecipitation. We demonstrated that DMBT1 was downregulated in OC cell lines, especially SKOV3 cells. Overexpression of DMBT1 significantly inhibited cell proliferation, colony formation, migration and invasion, as well as decreased Matrix Metalloproteinase‐2 (MMP‐2) and MMP‐7. DMBT1 caused a reduction of cell viability by treatment with cisplatin. Immunoprecipitation assay revealed a combination between DMBT1 and galectin‐3. DMBT1 could decrease the expression of galectin‐3 and inhibit the phosphorylation of PI3K and AKT, while overexpression of galectin‐3 reversed this effect. In summary, DMBT1 might inhibit the progression of OC and improve the sensitivity of SKOV3 cells to cisplatin through galectin‐3/PI3K/AKT pathway, giving a new insight into the role of DMBT1 in OC and enriching the potential strategies for OC treatment.Significance of the StudyThe present study focus on the role and the potential mechanism of DMBT1 in ovarian cancer (OC). We demonstrated that DMBT1 might inhibit the progression of ovarian by inhibiting cell proliferation, migration and invasion and increased the sensitivity to cisplatin through galectin‐3/PI3K/AKT pathway. The findings ensure the interaction relation between DMBT1 and galectin‐3 in OC, providing a novel biological marker for OC and enriching the potential strategies for OC treatment.

Upregulated CXCL14 is associated with poor survival outcomes and promotes ovarian cancer cells proliferation

Ovarian cancer is one of the common malignant tumours of female reproductive organs. Due to early diagnosis difficulties and lack of effective treatment in the late stage, ovarian cancer has the highest mortality rate in female reproductive system malignancies. Therefore, finding reliable early diagnosis indicators and new therapeutic targets for ovarian cancer is an urgent problem to be solved. Chemokine (C‐X‐C motif) ligand 14 (CXCL14) is a small cytokine belonging to the CXC chemokine family, which has been found to possess multi‐effects in tumourigenesis and development. Here, we reported that CXCL14 was preferentially expressed in ovarian cancer. By analysing the TCGA database, we found that CXCL14 was highly expressed in advanced ovarian cancer patients and correlated with poor prognosis. In addition, the abnormal high CXCL14 levels were observed in serum and ovarian tissue of ovarian cancer patients by qRT‐PCR and ELISA. In vitro and in vivo experiments both confirmed that overexpression of CXCL14 promoted the ovarian cancer cell proliferation. Moreover, transfection of CXCL14 increased the phosphorylation level of signal transducer and activator of transcription 3 (STAT3), and administration of STAT3 inhibitor III inhibited the tumour‐promoting effects of CXCL14. Therefore, our study suggests that CXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer.Significance paragraphCXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer.

A‐kinase‐interacting protein 1 promotes EMT and metastasis via PI3K/Akt/IKKβ pathway in cervical cancer

Overexpression of A‐kinase‐interacting protein 1 (AKIP1) has been reported in prostate and breast cancers. Nevertheless, the clinical potential of AKIP1 during the development of cervical cancer (CC) remains unclear. A series of experiments involving BdU, colony formation, wound healing and cell invasion assays were performed to determine cell proliferation, migration and invasion, respectively. Gene expression changes were validated by qRT‐PCR, Western blotting and immunocytochemistry. We found that AKIP1 expression is increased in CC cell lines and tissue specimens from CC patients. The elevated AKIP1 expression in primary tumours was related to lymph node metastasis in CC patients. In addition, we observed that overexpression of AKIP1 promotes CC cell proliferation. Enhanced expression of AKIP1 facilitated the migration and invasion of CC cells by inducing NF‐κB‐dependent epithelial‐mesenchymal transition (EMT). Moreover, mechanistic investigations revealed that AKIP1 induced nuclear translocation and phosphorylation of the p65 NF‐κB subunit through the PI3K/Akt/IKKβ pathway. Conversely, enhanced expression of phosphatase and tensin homologue (PTEN) inhibited this signalling pathway and restored an epithelial phenotype to CC cells in the presence of overexpressed AKIP1. Our results indicate that AKIP1 promotes the EMT and metastasis in CC by activating NF‐κB signalling through the PI3K/Akt/IKKβ pathway, suggesting that AKIP1 could be a pivotal regulator of an EMT axis in CC.Significance of the studyAKIP1 expression in the samples of CC patients and in in vitro tumour cell lines provides evidence that expression of AKIP1 in CC contributes to cancer progression. Our findings indicate that AKIP1 promotes the epithelial‐mesenchymal transition and metastasis in CC by activating NF‐κB signalling through the PI3K/Akt/IKKβ pathway, suggesting that AKIP1 is a pivotal regulator of an EMT axis in CC. AKIP1 could be implicated as a potential therapeutic target as well as a valuable biomarker for evaluating prognosis for patients with CC.

Evidence that cervical cancer cells cultured as tumorspheres maintain high CD73 expression and increase their protumor characteristics through TGF‐β production

Abstract Recently, a link between the biological activity of CD73 and tumorigenicity in solid tumors has been proposed. We previously reported that the generation of adenosine (Ado) by the activity of CD73 in cervical cancer (CC) cells induces transforming growth factor‐beta 1 (TGF‐β1) production to maintain CD73 expression. In the present study, we analyzed the participation of TGF‐β1 in CD73 expression and the development of protumoral characteristics in CaSki CC cells cultured as tumorspheres (CaSki‐T) and in monolayers (CaSki‐M). Compared with those in CaSki‐M cells, CD73 expression and Ado generation ability were significantly increased in CaSki‐T cells. CaSki‐T cells exhibited enrichment in the CSC‐like phenotype due to increases in the expression levels of stem cell markers (CD49f, CK17, and P63; OCT4 and SOX2), greater sphere formation efficiency (SFE), and an increase in the percentage of side population (SP) cells. Interestingly, compared with CaSki‐M cells, CaSki‐T cells produced a greater amount of TGF‐β1 and presented a marked protumor phenotype characterized by a significant decrease in the expression of major histocompatibility complex class‐I (MHC‐I) molecules, an increase in the expression of multidrug resistance protein‐I (MRP‐I) and vimentin, and an increase in the protein expression levels of Snail‐1 and Twist, which was strongly reversed with TGF‐β1 inhibition. These results suggest that the presence of TGF‐β1−CD73–Ado feedback loop can promote protumoral characteristics in the CC tumor microenvironment.