CaSki Cells Research Articles

Insights into the Mechanism of Curaxin CBL0137 Epigenetic Activity: The Induction of DNA Demethylation and the Suppression of BET Family Proteins.

The development of malignant tumors is caused by a complex combination of genetic mutations and epigenetic alterations, the latter of which are induced by either external environmental factors or signaling disruption following genetic mutations. Some types of cancer demonstrate a significant increase in epigenetic enzymes, and targeting these epigenetic alterations represents a compelling strategy to reverse cell transcriptome to the normal state, improving chemotherapy response. Curaxin CBL0137 is a new potent anticancer drug that has been shown to activate epigenetically silenced genes. However, its detailed effects on the enzymes of the epigenetic system of transcription regulation have not been studied. Here, we report that CBL0137 inhibits the expression of DNA methyltransferase DNMT3a in HeLa TI cells, both at the level of mRNA and protein, and it decreases the level of integral DNA methylation in Ca Ski cells. For the first time, it is shown that CBL0137 decreases the level of BET family proteins, BRD2, BRD3, and BRD4, the key participants in transcription elongation, followed by the corresponding gene expression enhancement. Furthermore, we demonstrate that CBL0137 does not affect the mechanisms of histone acetylation and methylation. The ability of CBL0137 to suppress DNMT3A and BET family proteins should be taken into consideration when combined chemotherapy is applied. Our data demonstrate the potential of CBL0137 to be used in the therapy of tumors with corresponding aberrant epigenetic profiles.

Fabrication of epidermal growth factor functionalized polymeric poly(lactic-co-glycolic acid) nanoparticles loaded SN-38 and perfluorocarbon for treatment and care: investigation of antiproliferative effects and apoptosis in cervical cancer cells

Patients with cervical cancer have a significant risk of tumor recurrence and metastasis. The discovery of effective treatments for cervical cancer is urgently needed. Poly(lactic-co-glycolic acid) (PLGA) was recently discovered to offer a promising therapeutic drug carrier. Hence, we developed dual-loaded PLGA nanoparticles (NPs) as a drug carrier to combat this problem with current chemotherapeutic drugs for cervical cancer. We engineered PLGA NPs with epidermal growth factor (EGF) functionalization and co-loaded them with SN-38 and perfluorocarbon (PC) to treat cervical cancer selectively. Cell counting kit-8 test results reveal that newly fabricated NPs effectively induce cell proliferation in cervical cancer cells. Further, flow cytometry, Hoechst 33342 staining and acridine orange and propidium iodide staining were used to determine the apoptosis of SN38/PC@EGF-PLGA NPs in HeLa and CaSki cells. And the release was pH-dependent when tested in vitro. Cervical cancer cells took up targeted SN38/PC@EGF-PLGA NPs at a greater rate than untargeted NPs. Furthermore, HeLa and CaSki cells were more sensitive to apoptosis induction and cell viability suppression when exposed to SN38/PC@EGF-PLGA NPs than nontargeted NPs. The findings of this study improve the exploration of SN38/PC@EGF-PLGA NPs in the new development of effective drug candidates for highly invasive cervical cancer in future.

Inhibitory effect of Lonicera japonica-derived exosomal miR2911 on human papilloma virus

Ethnopharmacological relevanceLonicera japonica Thunb. has been used as a traditional medicinal herb in China for thousands of years for its heat-clearing and detoxification effects. In recent years, experimental and clinical studies have shown that some Lonicera japonica-containing Chinese medicine prescriptions have been used to treat intraepithelia neoplasia caused by human papilloma virus (HPV) infection. However, its bioactive molecules and mechanism of action have not been fully explored. Aim of the studyIn this study, Lonicera japonica-derived exosomes was extracted and exosomal miR2911 was identified. Bioinformatic analysis indicated that miR2911 potentially binds to the sequence of HPV. The mechanism of miR2911 action on HPV and the effect of exosomal miR2911 on HPV-induced cervical cancer cells were investigated. MethodsThe potential targets of miR2911 on the HPV sequence were predicted and confirmed by using RNAhybrid and dual-luciferase reporter assays. Lonicera japonica exosomes were characterized by transmission electronic microscopy and zeta sizer analysis. RT-qPCR was used to measure miR2911 concentration in various tissues and exosomes. Synthetic miR2911 and GFP-E6/E7 plasmids were transfected into HEK293T cells to examine the effect of miR2911 on E6/E7 gene expression. The effects of miR2911 on endogenous E6/E7 mRNA and protein levels were detected in HPV16/18-positive cervical cancer cells by RT-qPCR and Western blotting. The proliferation and apoptosis of CaSki, SiHa and HeLa cells by the treatment of miR2911 or miR2911-containing exosomes were examined by CCK8, colony formation and flow cytometry assays. ResultsMiR2911 is found to be enriched in various Lonicera japonica tissues, and is stably present in Lonicera japonica-derived exosomes. It is observed that MiR2911 directly binds to E6 and E7 oncogenes of HPV16/18, leading to the suppression of their protein expression. In addition, the endogenous E6/E7 mRNA and protein levels were significantly decreased by using miR2911 treatment in HPV16/18-positive cervical cancer cells. Furthermore, both miR2911 and miR2911-containing exosomes inhibited cell proliferation of SiHa, CaSki and HeLa cells, meanwhile inducing the cell apoptosis through E6/E7-p53/Caspase3 axis. ConclusionOur findings indicate that miR2911, an active component present in Lonicera japonica exosomes, inhibits proliferation and induces apoptosis of cervical cancer cells by targeting the E6/E7 genes of HPV16/18. Thus, Lonicera japonica–derived exosomal miR2911 has implications for the development of novel therapeutic strategies for the treatment of HPV-associated cervical cancers.

TRIM21 promotes tumor progression and cancer stemness in cervical squamous cell carcinoma

BackgroundThe ubiquitin ligase family member triplex motif protein 21 (TRIM21), which is involved in the proliferation, metastasis, and selective death of tumor cells, is crucial in the ubiquitination of a number of tumor marker proteins. As research progresses, more studies demonstrate that TRIM21 expression levels can be used to predict cancer prognosis. However, it is unclear how exactly TRIM21 contributes to cervical squamous carcinoma. MethodsImmunohistochemistry, Western Blot, and q-PCR were utilized to determine the expression level of TRIM21 in 113 patients with CESC removed by stage I surgery at Xijing Hospital from 2018 to 2023 using paraffin-embedded tumor tissues and 12 pairs of fresh tumor tissues and their paracancerous tissues. Log-rank analysis using SPSS 23.0 was performed for prognosis and survival analysis using univariate/multifactorial analysis. CCK-8, wound-healing and Scratch assay verified that TRIM21 promoted cell proliferation, migration and invasion. The effect of overexpression and knockdown of TRIM21 on tumor stemness was examined using sphere-forming assay and Western Blot. Finally, we constructed a xenograft model to observe the effect of TRIM21 on tumorigenesis in Si Ha cell lines in vivo. ResultsTRIM21 expression is greater in CESC tissues than in paracancerous tissues, according to immunohistochemical data. Similarly, at the protein and mRNA levels, we verified this conclusion using Western-Blotting and q-PCR. Prognostic and OS analysis showed that TRIM21 expression levels are associated with individual prognostic factors. CCK-8, Wound healing, Transwell, and Sphere-forming tests all demonstrated that TRIM21 overexpression enhances Ca Ski cell proliferation, migration, invasion, and stemness. TRIM21 knockdown in Si Ha inhibited tumor cell proliferation, migration, invasion, and stemness. The experimental results of xenograft models demonstrated that TRIM21 knockdown in Si Ha cells inhibited tumor development. ConclusionTRIM21 is a poor predictor of prognosis for cervical squamous cell carcinoma and might open up new avenues for investigation into therapeutic targets.

PSME3 induces radioresistance and enhances aerobic glycolysis in cervical cancer by regulating PARP1

Cervical cancer (CC) ranks the fourth in gynecologic cancers. The incidence and mortality of CC has been decreased due to the cancer screening and early treatments in recent years, but the prognosis of CC patients at advanced stage is still sorrowful. Whether PSME3 exerted a role in the radioresistance of CC cells remains to be investigated. In this study, the expression of PSME3 in mRNA and protein levels was measured by RT-qPCR and western blot analysis, and increased expression of PSME3 in CC tissues and cells was observed. CCK-8 and colony formation assay revealed that the cell viability and proliferation of Hela and CaSki cells treated with different doses of X-ray was reduced due to the depletion of PSME3, indicating that silencing of PSME3 enhanced the radiosensitivity of CC cells. In addition, repair on DNA damage in CC cells was enhanced by PSME3 and the damage was attenuated by PSME3. Besides, the expression of glycolysis-related proteins (GLUT1, PGC-1α, LDHA and HK2) were enhanced by PSME3 but reduced by silencing PSME3 in CC cells. PSME3 restraint attenuated the levels of glucose consumption and lactate production, suggesting PSME3 depletion suppressed abnormal glycolysis of CC cells. Mechanically, PSME3 increased the PARP1 expression via elevating c-myc. Finally, we observed PSME3 attenuation inhibited CC growth in vivo. In conclusion, PSME3 enhanced radioresistance and aerobic glycolysis in CC by regulating PARP1, which might shed a light into the function of PSME3 in CC treatment.

A novel DNA damage repair-related gene signature predicting survival, immune infiltration and drug sensitivity in cervical cancer based on single cell sequencing.

Aberrant DNA damage repair (DDR) is one of the hallmarks of tumors, and therapeutic approaches targeting this feature are gaining increasing attention. This study aims to develop a signature of DDR-related genes to evaluate the prognosis of cervical cancer (CC). Differentially expressed genes were identified between high and low DDR groups of cells from the single-cell RNA sequencing dataset GSE168652 based on DDR scores. Using the ssGSEA and WGCNA methods, DDR-related differentially expressed genes were identified from different patients within the TCGA-CESC cohort. Using Cox analysis and LASSO regression analysis, a DDR-related gene signature was constructed based on the intersection of two groups of differentially expressed genes and DDR-related genes from WGCNA, and validated in GSE52903. Immune cell infiltration analysis, mutation analysis, survival analysis, drug sensitivity analysis, etc., were performed in different groups which were established based on the DDR gene signature scoring. A key gene affecting prognosis was selected and validated through biological experiments such as wound healing, migration, invasion, and comet assays. A novel DDR-related signature was constructed and the nomogram results showed this signature performed better in predicting prognosis than other clinical features for CC. The high DDR group exhibited poorer prognosis, weaker immune cell infiltration in the immune microenvironment, lower expression of immune checkpoint-related genes, lower gene mutation frequencies and more sensitivity to drugs such as BI.2536, Bleomycin and etc. ITGB1, ZC3H13, and TOMM20 were expressed at higher levels in CaSki and HeLa cells compared to ECT1 cells. Compared with the native CaSki and HeLa cells, the proliferation, migration, invasion and DDR capabilities of CaSki and HeLa cell lines with ITGB1 suppressed expression were significantly decreased. The 7 DDR-related gene signature was an independent and powerful prognostic biomarker that might effectively evaluate the prognosis of CC and provide supplementary information for a more personalized evaluation and precision therapy. ITGB1 was a potential candidate gene that may affect the DDR capacity of CC cells, and its mechanism of action was worth further in-depth study.

Butyrate inhibits the mitochondrial complex Ι to mediate mitochondria-dependent apoptosis of cervical cancer cells

BackgroundCervical cancer (CC) is a common gynecological malignancy with high morbidity worldwide. Butyrate, a short-chain fatty acid produced by intestinal flora, has been reported to inhibit cervical carcinogenesis. This study aimed to investigate the pro-apoptotic effects of butyrate on CC and the underlying mechanisms.MethodsHuman HeLa and Ca Ski cells were used in this study. Cell proliferation, cell migration and invasion were detected by CCK-8 and EdU staining, transwell and wound healing assay, respectively. Cell cycle, mitochondrial membrane potential and apoptosis were evaluated by flow cytometry. Western blot and RT-qPCR were carried out to examine the related genes and proteins to the mitochondrial complex Ι and apoptosis. Metabolite changes were analyzed by energy metabolomics and assay kits. The association between G protein-coupled receptor 41, 43, 109a and CC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA).ResultsCCK-8 results showed significant inhibition of CC cell proliferation induced by butyrate treatment, which was confirmed by EdU staining and cell cycle detection. Data from the transwell and wound healing assay revealed that CC cell migration was dramatically reduced following butyrate treatment. Additionally, invasiveness was also decreased by butyrate. Western blot analysis showed that cleaved Caspase 3 and cleaved PARP, the enforcers of apoptosis, were increased by butyrate treatment. The results of Annexin V/PI staining and TUNEL also showed an increase in butyrate-induced apoptotic cells. Expression of Cytochrome C (Cytc), Caspase 9, Bax, but not Caspase 12 or 8, were up-regulated under butyrate exposure. Mechanistically, the decrease in mitochondrial NADH and NAD + levels after treatment with butyrate was observed by energy metabolomics and the NAD+/NADH Assay Kit, similar to the effects of the complex Ι inhibitor rotenone. Western blot results also demonstrated that the constituent proteins of mitochondrial complex Ι were reduced by butyrate. Furthermore, mitochondria-dependent apoptosis has been shown to be initiated by inhibition of the complex Ι.ConclusionCollectively, our results revealed that butyrate inhibited the proliferation, migration and invasion of CC cells, and induced mitochondrial-dependent apoptosis by inhibiting mitochondrial complex Ι.

The effect of propofol on chemosensitivity of paclitaxel in cervical cancer cells.

Propofol is a drug with potential anticancer effect. This study aimed to explore the effect of propofol on chemosensitivity of cervical cancer cells to paclitaxel. HeLa and CaSki cells were selected for drug experiments. Cell viability was evaluated via CCK-8 assay, and the combination index (CI) was calculated by CompuSyn software. A clinically relevant concentration and IC30 of propofol were selected in combination with 5 nM paclitaxel. BrdU incorporation, transwell, and flow cytometry assays were utilized to evaluate cell proliferation, migration, invasion, and apoptosis. The expression of β-tubulin, stathmin 1, and GAPDH proteins was evaluated by Western blot. The stathmin 1 cDNA plasmid was used to establish stathmin 1-overexpressing CaSki cells. At clinically relevant concentrations (0-80 μM), propofol did not affect cancer cell viability, but high concentrations (100-800 μM) reduced cell viability. The CI values of propofol with IC30 (200 μM in HeLa; 400 μM in CaSki) combined with 5 nM paclitaxel were <1. The effect of propofol with IC30 combined with paclitaxel on cell proliferation, migration, invasion, and apoptosis were stronger than individual effect, while 30 μM propofol had no effect. The Western blot results showed 30 μM propofol did not affect β-tubulin and stathmin 1 expression in cells, although paclitaxel upregulated β-tubulin expression while downregulating stathmin 1 expression. Compared with paclitaxel alone, cotreatment with propofol at its IC30 and paclitaxel decreased stathmin 1 expression but had no effect on β-tubulin expression. High stathmin 1 expression weakened the effect of paclitaxel on cell viability and apoptosis, while propofol partially reversed these effect. Propofol at clinically relevant concentrations had no effect on the malignant biological behaviors of cervical cancer cells, while propofol at high concentrations decreased.Propofol with IC30 and paclitaxel had synergetic effect on cancer cells through a reduction in stathmin 1 expression.

Curcumin suppressed the proliferation and apoptosis of HPV-positive cervical cancer cells by directly targeting the E6 protein.

Most human papillomavirus (HPV) types, including HPV16 and HPV18, are closely related to the occurrence of cervical cancer, predominantly through the action of viral oncoproteins E6 and E7. Curcumin, the active ingredient of the turmeric plant, has been gaining attention over the past two decades as an antioxidant, anti-inflammatory, and anticancer agent. In the present study, the HPV-positive cervical cancer cells HeLa and CaSki were treated with curcumin, and the results showed that curcumin has a dose-dependent and time-dependent inhibitory effect on cell viability. In addition, apoptosis induction was further quantitatively confirmed through flow cytometric analysis. Furthermore, the influence of different concentrations of curcumin on the mitochondrial membrane potential was evaluated through JC-1 staining and found to dramatically decrease the membrane potential in treated HeLa and CaSki cells, suggesting the critical role of the mitochondrial pathway in their apoptosis-inducing effect. This study also demonstrated the wound-healing potential of curcumin, and the results of transwell assays showed that curcumin treatment inhibited HeLa and CaSki cell invasion and migration in a dose-dependent manner compared with the control treatment. Curcumin also downregulated the expression of Bcl-2, N-cadherin, and Vimentin and upregulated the expression of Bax, C-caspase-3, and E-cadherin in both cell lines. Further research showed that curcumin also selectively inhibited the expression of the viral oncoproteins E6 and E7, as demonstrated by western blot analysis; moreover, the downregulation of E6 was more significant than that of E7. Our research also showed that coculture with cells infected with siE6 lentivirus (siE6 cells) can inhibit the proliferation, invasion, and metastasis of HPV-positive cells. While the siE6 cells were also treated with curcumin, the effect of curcumin monotherapy was offset. In summary, our research shows that curcumin regulates the apoptosis, migration, and invasion of cervical cancer cells, and the mechanism may be related to its ability to downregulate E6. This study provides a foundation for future research on the prevention and treatment of cervical cancer.

RIPK1 and RIPK3 are positive prognosticators for cervical cancer patients and C2 ceramide can inhibit tumor cell proliferation in vitro.

The enzymes Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) und 3 (RIPK3) as well as the protein Mixed lineage kinase domain like pseudokinase (pMLKL) play a role in the signaling cascade of necroptosis. This is a form of programmed cell death which is caspase-independent. High-risk human papilloma virus infection can inhibit necroptosis. Thereby, a persistent infection and consequently the development of cervical cancer can be triggered. Aim of this study was the analysis of the expression of RIPK1, RIPK3 and pMLKL in cervical cancer tissue and the evaluation of its prognostic value on overall survival, progression-free survival and additional clinical parameters. The expression of RIPK1, RIPK3, and pMLKL in cervical cancer tissue microarrays of n = 250 patients was analyzed immunohistochemically. Further, the effect of C2 ceramide on several cervical cancer cell lines (CaSki, HeLa, SiHa) was examined. C2 ceramide is a biologically active short-chain ceramide that induces necroptosis in human luteal granulosa cells. Significantly longer overall survival and progression-free survival rates could be detected in cervical cancer patients expressing nuclear RIPK1 or RIPK3 alone or simultaneously (RIPK1 and RIPK3). Cell viability and proliferation was reduced through C2 ceramide stimulation of cervical cancer cells. Simultaneous stimulation of C2 ceramide and the pan-caspase inhibitor Z-VAD-fmk, or the RIPK1-inhibitor necrostatin-1, partly reversed the negative effect of C2 ceramide on cell viability. This observation could imply that caspase-dependent and -independent forms of cell death, including necroptosis, can occur. AnnexinV-FITC apoptosis staining induced a significant increase in apoptotic cells in CaSki and SiHa cells. The stimulation of CaSki cells with C2 ceramide led to a significant percentual increase in necrotic/intermediate (dying) cells after stimulation with C2 ceramide. In addition, after stimulation with C2 ceramide, CaSki and HeLa cells live cell imaging showed morphological changes which are common for necroptosis. In conclusion, RIPK1 and RIPK3 are independent positive predictors for overall survival and progression-free survival in cervical cancer patients. C2 ceramide can reduce cell viability and proliferation in cervical cancer cells by inducing most likely both apoptosis and necroptosis.

Extracellular vesicles derived from cervical cancer cells carrying MCM3AP-AS1 promote angiogenesis and tumor growth in cervical cancer via the miR-93/p21 axis

Tumor cells can promote angiogenesis by secreting extracellular vesicles (EVs). Meanwhile, tumor-derived EVs can carry long non-coding RNAs to activate pro-angiogenic signaling in endothelial cells. Here, we investigated the role of long non-coding RNA MCM3AP-AS1 carried by cervical cancer (CC) cell-derived EVs in the angiogenesis and the resultant tumor growth in CC, as well as the potential molecular mechanisms. LncRNAs significantly expressed in CC cell-derived EVs and CC were screened, followed by prediction of downstream target genes. EVs were isolated from HcerEpic and CaSki cell supernatants, followed by identification. The expression of MCM3AP-AS1 in CC was analyzed and its interaction with miR-93-p21 was confirmed. Following co-culture system, the role of MCM3AP-AS1 carried by EVs in HUVEC angiogenic ability, CC cell invasion and migration in vitro along with angiogenesis and tumorigenicity in vivo was assayed. MCM3AP-AS1 was overexpressed in CC cell-derived EVs as well as in CC tissues and cell lines. Cervical cancer cell-derived EVs could transfer MCM3AP-AS1 into HUVECs where MCM3AP-AS1 competitively bound to miR-93 and upregulate the expression of the miR-93 target p21 gene. Thus, MCM3AP-AS1 promoted angiogenesis of HUVECs. In the similar manner, MCM3AP-AS1 enhanced CC cell malignant properties. In nude mice, EVs-MCM3AP-AS1 induced angiogenesis and tumor growth. Overall, this study reveals that CC cell-derived EVs may transport MCM3AP-AS1 to promote angiogenesis and tumor growth in CC.

Asperuloside Promotes Apoptosis of Cervical Cancer Cells through Endoplasmic Reticulum Stress-Mitochondrial Pathway.

To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway. Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC50) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside. Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05). Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.

Abstract 2666: Preclinical investigations of concomitant tumor treating fields (TTFields) with cisplatin or paclitaxel for treatment of cervical cancer

Abstract Introduction: Cervical cancer is a serious health problem despite the fact it is highly preventable. Early-stage cervical cancer treatment often involves surgery; however advanced, recurrent, or metastatic cases require systemic therapy. Chemotherapy, mainly cisplatin, is the most commonly used systemic therapy for this cancer type. Combination regimens may be needed in the recurrent or metastatic settings, with first-line treatments including cisplatin with paclitaxel. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Concomitant treatment with TTFields and cisplatin or paclitaxel has shown benefit in other tumor types. In the current in vitro study, we tested the effectiveness of TTFields for the treatment of cervical cancer, and the possible benefit of applying TTFields together with first-line treatments for cervical cancer. Methods: Human cervical cancer cell lines - squamous cell carcinoma Ca Ski and SiHa cells, and adenocarcinoma HeLa cells - were treated with TTFields (72 h, 1 V/cm RMS) at frequencies of 100 to 400 kHz, and tested for cell count. For examining the efficacy of TTFields concomitant with cisplatin or paclitaxel, various doses of the drugs were applied together with TTFields (200 kHz), followed by measurements of cell count, colony formation, and apoptosis. Overall effect was defined as the product of percent reductions in cell count and colony formation. Results: TTFields treatment reduced cell count in all tested cervical cancer cell lines. 200 kHz were found to be effective and were used throughout the experiments. Dose response effects were seen with cisplatin or paclitaxel, and were augmented when TTFields were co-applied to the cells. Conclusions: These preclinical data suggest that TTFields may be an effective treatment against cervical cancer, and that applying them concomitantly with first-line treatment for this malignancy may provide enhanced effectiveness. Citation Format: Roni Frechtel-Gerzi, Daria Gerasimova, Einav Zeevi, Inbar Schlachet-Drukerman, Helena Mumblat, Antonia Martinez-Conde, Eyal Dor-On, Itai Tzchori, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti, Greg Palmer, Angeles A. Secord. Preclinical investigations of concomitant tumor treating fields (TTFields) with cisplatin or paclitaxel for treatment of cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2666.

Abstract 3779: Evaluation of anti-cancer efficacy of lipid nanoparticles containing siRNA against HPV16 E6 and E7 combined with cisplatin in xenograft model of cervical cancer

Abstract Background: High-risk human papillomavirus (HPV) type 16 is the major etiological agent found in more than 60% of patients with cervical cancer and associated with cancer development and progression. Persistent expression HPV E6 and E7 oncoproteins are essential for the initiation and maintenance of cervical cancer. The therapeutic approaches targeting HPV E6 and E7 have been proved to be highly efficient in killing malignant cells. The suppression of HPV 16 E6/E7 expression by the short interfering RNA (siRNA) was more effective in combination therapy with cisplatin (CDDP) for cervical cancer. ENB101-LNP is an ionizable lipid nanoparticles (LNPs) encapsulating siRNA against E6/E7 of HPV 16 for delivery to the tumor cells. Methods: To investigate the anticancer efficacy and mechanism of ENB101-LNP combined with cisplatin in cervical cancer; the xenograft model was generated by the injection of CaSki cells subcutaneously into BALB/c nude mice. Mice were randomly divided into six groups: 1) blank control; 2) 1mg/kg ENB101-LNP; 3) 3mg/kg ENB101-LNP; 4) CDDP; 5) 1mg/kg ENB101-LNP-CDDP; 6) 3mg/kg ENB101-LNP-CDDP. ENB101-LNP was treated with intravenous injection three times weekly for 3 weeks and CDDP was treated with IP injection once a week for 3 weeks. Tumor growth curves were plotted to calculate the tumor inhibition rate. Mice were sacrificed one week after the last treatment and the tumor tissues were investigated using histopathology, immunohistochemical staining and western blotting. Results: Compared with the control and the other treatment groups, the tumor growth was significantly inhibited (P &amp;lt; 0.05). The tumor inhibition rate was 29.7% in 1mg/kg ENB101-LNP group, 29.6% in 3mg/kg ENB101-LNP group, 34.0% in CDDP group, 47.0% in 1mg/kg ENB101-LNP-CDDP group. At the dose of 3mg ENB101-LNP/kg combined with CDDP exhibited superior antitumor efficacy, the tumor inhibition rate was 68.8%. The successful ENB101-LNP mediated knockdown (with up to 80%) of HPV16 E6/E7 in tumors of both 1mg/kg and 3mg/kg groups was confirmed by RT-PCR. The ENB101-LNP increased p53 and p21 expression in tumors compared to control and CDDP alone groups and combination treatment with CDDP showed decreased expression of PD-L1 expression compared to CDDP alone group. The treatment of ENB101-LNP and combination with CDDP showed significant increase in apoptotic cells compared to control and single-agent groups respectively. Conclusions: The ENB101-LNP inhibiting E6 and E7 of HPV 16 in combination with CDDP showed promising anticancer activity in the cervical cancer cells xenograft mouse model. Citation Format: Sung Wan Kang, Shin-Wha Lee, Ji-young Lee, Ok Ju Kang, Hyejeong Kim, Yisak Kim, Yong-Man Kim. Evaluation of anti-cancer efficacy of lipid nanoparticles containing siRNA against HPV16 E6 and E7 combined with cisplatin in xenograft model of cervical cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3779.

HBXIP knockdown inhibits FHL2 to promote cycle arrest and suppress cervical cancer cell proliferation, invasion and migration.

Hepatitis B X-interacting protein (HBXIP) and four and a half LIM domain 2 (FHL2) have been reported to serve as independent biomarkers for cervical cancer. The present study evaluated the effects of HBXIP on cervical cancer in terms of its cellular malignant characteristics. Reverse transcription-quantitative PCR and western blotting were used to assess the mRNA and protein expression levels of HBXIP and FHL2 in the human endocervical epithelial End1/E6E7 cell line and the cervical cancer HeLa, CaSki, C33A and SiHa cell lines. After knocking down HBXIP expression by transfection of small interfering RNAs targeting HBXIP, cell cycle progression was assessed using flow cytometry with PI staining. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, wound healing and Transwell assays were used to assess cell proliferation, migration and invasion, respectively. Furthermore, co-immunoprecipitation assay was used to evaluate the potential binding relationship between HBXIP and FHL2. Western blotting was used for the analysis of HBXIP and FHL2, cell cycle-associated proteins, including cyclin D1 and cyclin D2, metastasis-associated proteins, including MMP2 and MMP9, and Wnt/β-catenin signaling-associated proteins, including β-catenin and c-Myc. Both HBXIP and FHL2 were found to be highly expressed in cervical cancer cells compared with that in the human endocervical epithelial cell line. HBXIP knockdown suppressed the proliferation, invasion and migration of HeLa cells, but promoted cell cycle arrest at the G0/G1 phase. HBXIP was demonstrated to interact with FHL2, and HBXIP knockdown also inhibited FHL2 mRNA and protein expression. By contrast, FHL2 overexpression reversed the inhibitory effects of HBXIP knockdown on the malignant characteristics of cervical cancer cells. Furthermore, HBXIP knockdown blocked the Wnt/β-catenin signaling pathway in HeLa cells, which was also partially reversed by FHL2 overexpression; the decreased β-catenin and c-Myc expression caused by HBXIP knockdown was increased again after FHL2 was overexpressed. In conclusion, these results suggest that HBXIP knockdown suppressed the malignant characteristics of cervical cancer cells through the downregulation of FHL2 expression, indicating a promising insight into the therapeutic target of cervical cancer.

The hsa_circ_0000276-ceRNA regulatory network and immune infiltration in cervical cancer

BackgroundOur previous studies have confirmed that miR-154-5p can regulate pRb expression, and thus, play a tumor suppressor role in HPV16 E7-induced cervical cancer. However, its upstream molecules have not been elucidated in the progression of cervical cancer. This study aimed to explore the role of the miR-154-5p upstream molecule, hsa_circ_0000276 in cervical cancer development and its possible mechanisms of action.MethodsWe detected differences in whole transcriptome expression profiles of cervical squamous carcinoma and tissues adjacent to cervical cancer tissues from patients using microarray technology to predict circular RNAs (circRNAs) with binding sites to miR-154-5p. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of hsa_circ_0000276 (which had the strongest binding capacity to miR-154 and was selected as the target molecule) in cervical cancer tissues, followed by in vitro functional assays. Downstream microRNAs (miRNAs) and mRNAs of hsa_circ_0000276 were identified using transcriptome microarray data and databases, while the protein–protein interaction networks were obtained using STRING. A competing endogenous RNA (ceRNA) network centered on hsa_circ_0000276 was constructed using Cytoscape and GO and KEGG databases. Abnormal expression and prognosis of critical downstream molecules were analyzed using gene databases and molecular experiments. qRT-PCR and western blot analysis was performed to verify the expression of candidate genes.ResultsWe identified 4,001 differentially expressed circRNAs between HPV16-positive cervical squamous carcinoma and benign cervical tissues and 760 circRNAs targeting miR-154-5p, including hsa_circ_0000276. hsa_circ_0000276 and miR-154-5p directly bound, and hsa_circ_0000276 was upregulated, in cervical precancerous lesions and cervical cancer tissues and cells. Silencing hsa_circ_0000276 inhibited G1/S transition and cell proliferation and promoted apoptosis in SiHa and CaSki cells. Bioinformatics analysis showed that the hsa_circ_0000276 ceRNA network included 17 miRNAs and seven mRNAs, and downstream molecules of hsa_circ_0000276 were upregulated in cervical cancer tissues. These downstream molecules were associated with a poor prognosis and affected cervical cancer-associated immune infiltration. Of these, expression of CD47, LDHA, PDIA3, and SLC16A1 was downregulated in sh_hsa_circ_0000276 cells.ConclusionsOur findings show that hsa_circ_0000276 exerts cancer-promoting effects in cervical cancer and is an underlying biomarker for cervical squamous cell carcinoma.

The HPV16 E6, E7/miR-23b-3p/ICAT signaling axis promotes proliferation, migration, invasion and EMT of cervical cancer cells.

Cervical cancer (CC) remains one of the most common female malignancies, with higher incidence and mortality rates. more than 99% of CCs are associated with persistent infection with high-risk human papillomavirus. In view of the growing evidence that HPV 16 E6 and E7, two key oncoproteins encoded by HPV 16, regulate the expression of many other multifunctional genes and downstream effectors that contribute to the development of CC. Herein, we undertook a comprehensive effort into how HPV16 E6, E7 oncogenes affect the progression of CC cells. Previous studies have shown that ICAT expression was significantly increased in CC and had a pro-cancer effect. We observed that knockdown of HPV16 E6, E7 expression in SiHa and CasKi cells resulted in significant inhibition of ICAT expression and upregulation of miR-23b-3p expression. Besides, dual luciferase assays confirmed that ICAT was a target gene of miR-23b-3p, and negatively modulated by miR-23b-3p. Functional experiments showed that the overexpression of miR-23b-3p suppressed malignant behaviors of CC cells, such as migration, invasion and EMT. The overexpression of ICAT counteracted the suppressive effect of miR-23b-3p on HPV16-positive CC cells. Furthermore, after the knockdown of HPV16 E6 and E7, the inhibition of miR-23b-3p could increase the ICAT expression and rescue the siRNA HPV16 E6, E7-mediated suppressive impact on the aggressiveness of SiHa and CaSki cells. Collectively, our findings uncover that HPV16 E6, E7/miR-23b-3p/ ICAT axis plays an important role in HPV16-positive CC pathogenesis, which may serve as a promising therapeutic target for HPV16-associated CC.

Enhancing autophagy leads to increased cell death in radiation-treated cervical cancer cells

This study was carried out to determine the effect of autophagy modulation in radiation treatment of cervical cancer cells. HeLa and CaSki cells were irradiated with γ-rays (2 Gy/min) after treatment with an autophagy inducer (rapamycin) and inhibitor (3-MA). Expression of LC3 and cell death in two cell preparations were examined. In addition, expression of Caspase-3 and PARP were examined after radiation alone and with autophagy inhibitor treatment. A notable increment of LC3 expression was detected after radiation in both cell lines. Cell viability was observed to decrease in 3-MA-treated cells compared to radiation alone, and even further in rapamycin-treated cells. Apoptosis was confirmed to occur later than autophagy in radiation treatment, and inhibition of autophagy derived a decrease in apoptosis. In conclusion, radiation-induced autophagy may be regulated by modulators, and autophagy augmentation yields an increase in cervical cancer cell death under radiation. Impact statement What is already known on this subject? Autophagy is known to contribute both to tumour cell survival and death against radiation therapy. The effect of induction or inhibition of radiation-induced autophagy on cervical cancer cell death is not clear. What the results of this study add? Cell viability was observed to decrease in 3-MA-treated cells compared to radiation alone, and even further in rapamycin-treated cells. Apoptosis occurred later than autophagy in radiation treatment, and inhibition of autophagy derived a decrease in apoptosis. What the implications are of these findings for clinical practice and/or further research? Our results suggest that radiation-induced autophagy may be regulated by modulators, and autophagy augmentation yields an increase in cervical cancer cell death under radiation.

The Phytochemical α-Mangostin Inhibits Cervical Cancer Cell Proliferation and Tumor Growth by Downregulating E6/E7-HPV Oncogenes and KCNH1 Gene Expression.

Cervical cancer is the fourth most common cancer among women worldwide. The main factor associated with the onset and progression of this neoplasia is the human papillomavirus (HPV) infection. The HPV-oncogenes E6 and E7 are critical drivers of cellular transformation, promoting the expression of oncogenes such as KCNH1. The phytochemical α-mangostin (AM) is a potent antineoplastic and antiviral compound. However, its effects on HPV oncogenes and KCNH1 gene expression remain unknown. This study evaluated the effects of AM on cell proliferation, cell cycle distribution and gene expression, including its effects on tumor growth in xenografted mice. AM inhibited cell proliferation in a concentration-dependent manner, being the most sensitive cell lines those with the highest number of HPV16 copies. In addition, AM promoted G1-cell cycle arrest in CaSki cells, while led to cell death in SiHa and HeLa cells. Of interest was the finding of an AM-dependent decreased gene expression of E6, E7 and KCNH1 both in vitro and in vivo, as well as the modulation of cytokine expression, Ki-67, and tumor growth inhibition. On these bases, we suggest that AM represents a good option as an adjuvant for the treatment and prevention of cervical cancer.

A 640 × 640 ISFET array for detecting cell metabolism

Ion sensitive field effect transistor (ISFET) devices are highly accurate, convenient, fast and low-cost in the detection of ions and biological macromolecules, such as DNA molecules, antibodies, enzymatic substrates and cellular metabolites. For high-throughput cell metabolism detection, we successfully designed a very large-scale biomedical sensing application specific integrated circuit (ASIC) with a 640 × 640 ISFET array. The circuit design is highly integrated by compressing the size of a pixel to 7.4 × 7.4 μm2 and arranging the layout of even and odd columns in an interdigital pattern to maximize the utilization of space. The chip can operate at a speed of 2.083M pixels/s and the dynamic process of the fluid flow on the surface of the array was monitored through ion imaging. The pH sensitivity is 33 ± 4 mV/pH and the drift rate is 0.06 mV/min after 5 h, indicating the stability and robustness of the chip. Moreover, the chip was applied to monitor pH changes in CaSki cells metabolism, with pH shifting from 8.04 to 7.40 on average. This platform has the potential for continuous and parallel monitoring of cell metabolism in single-cell culture arrays.