Evaluation of anti-cancer potential of Excoecaria agallocha (L.) leaf extract on human cervical cancer (SiHa) cell line and assessing the underlying mechanism of action

Background The incidence of cervical cancer is increasing at an alarming rate in many countries and presently, it is the most common form of malignant cancer being reported among women in India. Development of novel approach for cervical cancer therapy, sparing healthy normal cells overcoming the limitations of prevailing therapies is of prime importance. Mangroves constitute a significant repository of medicinally important plants. Thus, in this study, we aimed to determine the anticancer activity of the mangrove Excoecaria agallocha L. leaf extracts on human cervical cancer (SiHa HPV 16+) cell line with subsequent characterization of the bioactive compounds conferring the anticancer activity and studying the probable underlying mechanism of action of the purified plant extract. Results The plant extract was subjected to silica gel column chromatography and the fractions obtained were analyzed for cytotoxic activity against SiHa cells by MTT assay. One out of the three eluted fractions exhibited selective toxicity against SiHa cells with an IC50 value of 15.538 ± 0.577 µg/mL, while it had no cytotoxic effect on normal healthy human peripheral blood mononuclear cells. High-resolution liquid chromatography mass spectroscopy, coupled to electron spray ionization and diode array detection analysis, led to the structure elucidation and identification of a few pharmacologically important compounds, with Bergenin being present in the highest abundance. Fluorescence microscopy results revealed that the plant extract fraction induced LC3 puncta formation, in EGFP- SiHa cells indicating the onset of autophagy, with simultaneous stimulation of mitophagy. The plant extract also inhibited proliferation of the SiHa-smac-mCherry cells by second mitochondria-derived activator of caspase (SMAC)—induced cytochrome c dependent apoptosis, that was further confirmed with Caspase-3 activation by colorimetric assay. The GFP-dgn in SiHa cells was remarkably protected from proteasomal degradation that might upregulate the survivability of the cells significantly. Flow cytometry followed by Western blot analysis further asserted the ability of the plant extract fraction to cause cell cycle arrest of SiHa cells in the G2/M phase by significantly reducing protein expression levels of cyclin B1 and D1, decreasing Cdc2 level and simultaneously increasing p21 and p53 levels. Conclusion It could be inferred that the aqueous extract of E. agallocha successfully decreased the proliferation of SiHa cervical cancer cells through induction of autophagy and apoptosis in a concerted manner, with simultaneous stimulation of mitophagy and G2/M phase cell cycle arrest, hinting at Bergenin being the major compound conferring the anti-cancer activity of the plant extract. Thus, isolation of the identified bioactive compounds from E. agallocha and their subsequent purification for drug development might serve as a novel medicinal approach for the treatment of cervical cancer in conjugation with existing therapeutic methods.

CircCASC15-miR-100-mTOR may Influence the Cervical Cancer Radioresistance

Background Cervical cancer has ranked the top one in gynecological malignancies for incidence. Radioresistance is now becoming a leading reason of recurrence. Methods Our microRNA array data indicated that the miRNA-100 level decreased significantly during radioresistance. In this study, we up-regulated miR-100 in Hela and Siha cells by using miR-100 mimics and observed apoptosis, proliferation, cell cycle and invasion. Results It turned out that with overexpression of miR-100, the cells had more apoptosis and less invasiveness as well as proliferation. It may also influence cell cycle via target gene mTOR, and it deed reduced EMT. To examine the role of miR-100 in radioresistance, there was no significant result showed by BSP.While the circCASC15 has been identified with sponge function according to RNA pull down and ISH. Conclusion The conclusions indicate miR-100 is a tumor suppressor gene and could be a therapeutic target in radio-resistant cervical cancers.

The degradation of Rap1GAP via E6AP-mediated ubiquitin-proteasome pathway is associated with HPV16/18-infection in cervical cancer cells

Background Cervical cancers are closely associated with persistent high-risk human papillomaviruses (HR HPV) infection. The main mechanism involves the targeting of tumor suppressors, such as p53 and pRB, for degradation by HR HPV-encoded oncoproteins, thereby leading to tumorigenesis. Rap1GAP, a tumor suppressor gene, is down-regulated in many cancers. Previous studies have revealed that down-regulation of Rap1GAP is correlated with HPV16/18 infection in cervical cancer. However, the molecular mechanism remains unclear. In this study, we aimed to address the degradation pathway of Rap1GAP in HPV-positive cervical cancer cells. Methods HPV-positive (HeLa and SiHa) and negative (C33A) cervical cancer cells were used to analyze the pathways of Rap1GAP degradation. MG132 (carbobenzoxy-leucyl-leucyl-leucine) was used to inhibit protein degradation by proteasome. Co-immunoprecipitation (co-IP) was used to detect the interaction between Rap1GAP and E6AP. siRNA for E6AP was used to silence the expression of E6AP. Rapamycin was used to induce cell autophagy. Western blotting was used to check the levels of proteins. Results Following treatment with MG132, the levels of Rap1GAP were increased in the HR HPV-positive HeLa and SiHa cells, but not in the HPV-negative C33A cells. Co-immunoprecipitation assay revealed ubiquitinated Rap1GAP protein in HeLa and SiHa cells, but not in C33A cells. E6-associated protein (E6AP) mediated the ubiquitination of Rap1GAP by binding to it in HeLa and SiHa cells, but not in C33A cells. However, the levels of Rap1GAP were decreased in HeLa and SiHa cells after knocking down E6AP by siRNA. Silencing of E6AP did not affect the levels of Rap1GAP in C33A cells. Autophagy marker p62 was decreased and LC3 II/LC3 I was increased after knocking down E6AP in HeLa cells, but not in C33A cells. The levels of Rap1GAP were decreased after treating the cells with rapamycin to induce cell autophagy in HeLa and C33A cells. Conclusion Rap1GAP may be degraded by autophagy in cervical cancer cells, but HPV infection can switch the degradation pathway from autophagy to E6AP-mediated ubiquitin-proteasome degradation. E6AP may be a key component of the switch.

Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER–mitochondria crosstalk in SiHa cells

Background Trichomonas vaginalis causes lesions on the cervicovaginal mucosa in women; however, its pathogenesis remains unclear. We have investigated the involvement of the endoplasmic reticulum (ER) in the induction of apoptosis by T. vaginalis and its molecular mechanisms in human cervical cancer SiHa cells. Methods Apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response and Bcl-2 family protein expression were evaluated using immunocytochemistry, flow cytometry, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide dye staining and western blotting. Results Trichomonas vaginalis induced mitochondrial ROS production, apoptosis, the ER stress response and mitochondrial dysfunction, such as MMP depolarization and an imbalance in Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA; ER stress inhibitor) significantly alleviated apoptosis, mitochondrial ROS production, mitochondrial dysfunction and ER stress response in a dose-dependent manner. In addition, T. vaginalis induced the phosphorylation of apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinases (JNK) in SiHa cells, whereas 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis and ER stress response in SiHa cells, in a dose-dependent manner. Furthermore, T. vaginalis excretory/secretory products also induced mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells, in a time-dependent manner. Conclusions Trichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress responses, and also promotes ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells. These data suggest that T. vaginalis-induced apoptosis is affected by ROS and ER stress response via ER–mitochondria crosstalk. Graphical Abstract

4SC-202 Exerts An Anti-tumor Effect in Cervical Cancer By Targeting PRLR Signaling Pathway

The aim of the present study is to investigate whether 4SC-202, a selective class I histone deacetylase inhibitor (HDACi), plays an anti-tumor role in cervical cancer (CC) by targeting prolactin receptor (PRLR). CCK-8 and colony formation assays were used to evaluate the effects of 4SC-202 on the proliferation of CC cells in vitro. Effects of 4SC-202 on the cell cycle distribution and apoptosis in SiHa cells were determined by flow cytometry and western blotting, respectively. Immunofluorescence and western blotting were performed to detect the activities of PRLR-related pathways and PRLR expression in CC cells. A xenograft tumor model in nude mice was established to examine effects of 4SC-202 on the tumor growth, apoptosis and PRLR-related pathways in vivo. The biochemical analyzer and H&E staining were used to detect the serum biochemical indexes and organ toxicity. 4SC-202 inhibited the proliferation of CC cells (SiHa, HeLa, and CaSki) in vitro in a time- and dose-dependent manner. SiHa cells were treated with 1 or 5 μM 4SC-202 for 72 h and then subjected to various functional assays. The assays showed that 4SC-202 significantly induced G2/M phase arrest and apoptosis, while inhibiting the activities of PRLR-related pathways and PRLR expression. In addition, 4SC-202 reduced tumor growth and induced apoptosis in vivo. 4SC-202 down-regulated the expression of PRLR and activities of PRLR-related pathways in the mouse model, displayed no effects on serum biochemical indicators and caused no toxicity to mouse organs. This finding suggests that 4SC-202 may serve as a novel therapeutic agent for CC.

SIRT1 promotes proliferation, migration, and invasion of cervical cancer cell and is upregulated by human papillomavirus 16 E7 oncoprotein

SIRT1 (silent information regulator 1), a NAD+-dependent III class histone deacetylase, plays crucial roles in cell proliferation, apoptosis, senescence, metabolism, and stress responses. Nevertheless, the role of SIRT1 in tumorigenesis remains unclear. In the present study, we measured expression levels of SIRT1 and HPV16 E7 protein in cervical cancer tissue and calculated their correlations. We measured the effect of silencing SIRT1 on the proliferation, migration, invasion, and apoptosis in human cervical cancer SiHa cells. Immunohistochemistry results revealed that the expression of SIRT1 was upregulated with progression from CINII-III to cervical cancer, but was not expressed in normal cervical tissues and CINI. There was a positive correlation between SIRT1 expression and HPV16 E7 expression in cervical cancer tissues, and silencing of HPV16 E7 downregulated the expression of SIRT1. Depletion of SIRT1 significantly downregulated SIRT1 expression, and inhibited proliferation, migration, and invasion of SiHa cells, inducing apoptosis. Taken together, the data suggest that SIRT1 promotes cervical cancer carcinogenesis. SIRT1 inhibition is a potential treatment strategy for cervical cancer.

Antitumor activity of gambogic acid lysinate in human SiHa cells in vitro and in vivo

Background: Cervical cancer is a major cause of death for women worldwide and human papillomavirus (HPV) infection is the main cause of cervical cancer. The purpose of this study was to explore the anti-tumor activity of gambogic acid lysinate and clarify its mechanism in SiHa cells. Methods: In the present study, cell viability was detected by means of an MTT assay, a cell growth curve was drawn with Microsoft Excel 2010, the cell cycle and cell apoptosis were evaluated by flow cytometry, Western blotting was employed to explore the mechanism of gambogic acid lysinate, and caspase-3 activity was determined with a colorimetric Caspase-3 assay kit. Additionally, the in vivo antitumor activity of gambogic acid lysinate was studied through a xenograft tumor model established with nude mice. Results: The results showed that gambogic acid lysinate inhibited the proliferation of both SiHa cells (half-maximal inhibitory concentration (IC50) values: 0.83 μmol/l and 0.77 μmol/l for 48 h and 72 h) and HeLa cells (IC50 >2 μmol/l). In SiHa cells, gambogic acid lysinate (1 and 2 μmol/l) inhibited cell proliferation and 2 μmol/l gambogic acid lysinate induced cell apoptosis and decreased the number of S phase cells. Both 1 and 2 μmol/l gambogic acid lysinate increased the number of G0/G1 phase cells. The results of a Western blot assay demonstrated that P53 and P21 were involved in SiHa cell G0/G1 phase arrest and that Bcl-2 and BAX were involved in SiHa cell apoptosis. An in vivo study showed that the growth of SiHa cell xenograft tumors was inhibited by gambogic acid lysinate (2.5 mg/kg body weight), however, gambogic acid lysinate (2.5 mg/kg body weight) had no significant effect on mouse weight gain. Conclusions: gambogic acid lysinate is a promising candidate for cervical cancer therapy.

Role and Mechanism of Exosome CircRNA Eukaryotic Translation Initiation Factor 4γ2 (EIF4G2) in Cervical Cancer

Our work was to evaluate Exosome CircRNA EIF4G2 in cervical cancer development. Methods: Using Hela and Siha in our present study. Transfection vector, exosome cirEIF4G2, exosome si-NC or exosome si-circEIF4G2 in cells. Using RT-qPCR to measure circEIF4G2 gene expression in difference cell groups. Evaluating cell proliferation, apoptosis, invasion and wound healing rate by MTT, flow cytometry, transwell and wound healing assay. The relative proteins, HPV16 E6 and HPV16 E7, were evaluated by WB assay. With Exosome CircRNA EIF4G2 transfection, Hela and Siha cells proliferation, invasion cells number and wound healing rates were significantly increased and cells apoptosis were significantly depressed (P < 0.001, respectively) with HPV16 E6 and HPV16 E7 proteins expression were significantly up-regulation (P < 0.001, respectively). However, with Exosome si-CircRNA EIF4G2 transfection, Hela and Siha cells proliferation, invasion cells number and wound healing rates were significantly depressed and cells apoptosis were significantly increased (P < 0.001, respectively) with HPV16 E6 and HPV16 E7 proteins expression were significantly down-regulation (P < 0.001, respectively). Exosome CircRNA EIF4G2 as an oncology role in cervical cancer via regulation HPV16 E6/E7 up-regulation in vitro study.

Hsp90 up-regulates PD-L1 to promote HPV-positive cervical cancer via HER2/PI3K/AKT pathway

Background HPV16 is the predominant cancer-causing strain that is responsible for over 50% of all cervical cancers. In this study, we aim to investigate the therapeutic effect of heat shock protein 90 (Hsp90) knockdown on HPV16+ cervical cancer progression and the underlying mechanism. Methods The transcript and protein expression of Hsp90 in normal cervical and HPV16+ cervical cancer tissues and cell lines were detected by qRT-PCR, immunohistochemistry staining and Western blot. Hsp90 knockdown clones were established using HPV16+ cervical cancer cell line Caski and SiHa cells. The effect of Hsp90 knockdown on HER2/PI3K/AKT pathway and PD-L1 expression was characterized using qRT-PCR and Western blot analysis. Cell proliferation and migration were determined using MTT and transwell assays. Using mouse xenograft tumor model, the impact of Hsp90 knockdown and PD-L1 overexpression on tumor progression was evaluated. Results Hsp90 expression was up-regulated in HPV16+ cervical cancer tissues and cells. Knockdown of Hsp90 inhibited proliferation and migration of Caski and SiHa cells. PD-L1 expression in cervical cancer tissues was positively correlated with Hsp90 expression, and Hsp90 regulated PD-L1 expression via HER2/PI3K/AKT signaling pathway. The results of mouse xenograft tumor model demonstrated Hsp90 knockdown suppressed tumor formation and overexpression of PD-L1 simultaneously eliminated the cancer-suppressive effect of Hsp90 knockdown. Conclusion In this study, we demonstrated a promising tumor-suppressive effect of Hsp90 knockdown in HPV16+ cervical cancers, and investigated the underlying molecular pathway. Our results suggested that Hsp90 knockdown holds great therapeutic potential in treating HPV16+ cervical cancers.