Applied Biochemistry and Biotechnology

Screening and Identification of Potential iNOS Inhibitors to Curtail Cervical Cancer Progression: an In Silico Drug Repurposing Approach

Cervical cancer is the second most common cause of cancer deaths in women worldwide and remains the main reason of mortality among women of reproductive age in developing countries. Nitric oxide is involved in several physiological functions inclusive of inflammatory and immune responses. However, the function of NO in tumor biology is debatable. The inducible NOS (iNOS/NOS2) isoform is the one responsible to maintain the levels of NO, and it exhibits pleotropic effects in various cancers with concentration-dependent pro- and anti-tumor effects. iNOS triggers angiogenesis and endothelial cell migration in tumors by regulating the levels of vascular endothelial growth factor (VEGF). In drug discovery, drug repurposing involves investigations of approved drug candidates to treat various other diseases. In this study, we used anti-cancer drugs and small molecules to target iNOS and identify a potential selective iNOS inhibitor. The structures of ligands were geometrically optimized and energy minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker, and ligands were selected based on MolDock score, Rerank score, and H-bonding energy. In the study shown, venetoclax compound demonstrated excellent binding affinity to iNOS protein. This compound exhibited the lowest MolDock score and Rerank score with better H-bonding energy to iNOS. The binding efficacy of venetoclax was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions. Based on the results, venetoclax emerges to be a promising potential iNOS inhibitor to curtail cervical cancer progression.

Synthesis of Bimetallic Palladium/Zinc Oxide Nanocomposites Using Crocus sativus and Its Anticancer Activity via the Induction of Apoptosis in Cervical Cancer

Palladium (Pd) and zinc oxide (ZnO) (Pd/ZnO NPs) bimettalic nanocomposites still lag much too far behind other nanoparticles investigated for various biological uses in the area of cancer treatments. Chemically created nanoparticles agglomerate under physiological conditions, impeding their use in biomedical applications. In this study, a straightforward and environmentally friendly method for creating bimetallic nanoparticles (NPs) by combining palladium (Pd) and zinc oxide (ZnO) using Crocus sativus extract (CS-Pd/ZnO NCs) was reported; the bio-synthesize bimetallic palladium/zinc oxide nanocomposites and their antioxidant and anti-cancer properties were assessed. The developed Pd/ZnO NPs were characterized using different approaches, including UV-vis, DLS, FTIR, EDX, and SEM analyses. The present investigation shows how nanocomposites are made, their distinctive properties, antioxidant activity, anticancer mechanisms, and their potential therapeutic applications. DPPH and ABTS tests were used to investigate antioxidant activity. Further, the effects of CS-Pd/ZnO NCs on HeLa cells were assessed using the cell viability, ROS generation, MMP levels, and induced apoptosis. Apoptosis induction was measured using an Annexin V-fluorescein isothicyanate assay. Cell DNA was stained with propidium iodide to evaluate the impact upon this cell cycle. Time-dependent cell death was carried on by CS-Pd/ZnO NCs. The maximum inhibitory effect was 59 ± 3.2 when dosages of 4.5 µg/mL or higher were delivered after 24 h of treatment. Additionally, the CS-Pd/ZnO NCs caused HeLa cells to undergo apoptosis. Apoptotic HeLa cells were present in 35.64% of the treated cells at 4.5 µg/mL, and the cell cycle arrest at G0/G1 phase occurred concurrently. According to these findings, the CS-Pd/ZnO NCs may be a promising candidate for the creation of brand-new cervical cancer treatment.

Combination of Formononetin and Sulforaphane Natural Drug Repress the Proliferation of Cervical Cancer Cells via Impeding PI3K/AKT/mTOR Pathway

Natural substances have been demonstrated to be an unrivalled source of anticancer drugs in the present era of pharmacological development. Plant-based substances, together with their derivatives through analogues, play a significant character in the treatment of cancer by altering the tumor microenvironment and several signaling pathways. In this study, it was investigated whether the natural drugs, formononetin (FN) and sulforaphane (SFN), when combined, assess the efficacy of inhibiting cervical cancer cell proliferation by impeding the PI3K/Akt/mTOR signaling pathway in HeLa cells. The cells were treated with the combination of FN and SFN (FN + SFN) in various concentrations (0-50 µM) for 24 h and then analyzed for various experiments. The combination of FN + SFN-mediated cytotoxicity was analyzed by MTT assay. DCFH-DA staining was used to assess the ROS measurement, and apoptotic changes were studied by dual (AO/EtBr) staining assays. Protein expressions of cell survival, cell cycle, proliferation, and apoptosis protein were evaluated by flow cytometry and western blotting. Results showed that the cytotoxicity of FN and SFN was determined to be around 23.7 µM and 26.92 µM, respectively. Combining FN and SFN causes considerable cytotoxicity in HeLa cells, with an IC

Analysis of Correlation Between LncRNA TDRG1 Expression and its Prognosis in Cervical Carcinoma Tissues

To explore and analyze the correlation between LncRNA TDRG1 expression degree and the prognosis of cervical carcinoma tissues. The cervical cancer tissues and para-carcinoma tissues of 106 patients with cervical carcinoma surgically removed in our hospital were chosen as specimens. LncRNA TDRG1 expression in cervical carcinoma tissues and para-carcinoma tissues was inspected by real-time fluorescence quantitative PCR, and the correlation between LncRNA TDRG1 and the clinicopathological parameters and disease prognosis was analyzed. The relative expression of LncRNA TDRG1 in cervical carcinoma tissues was critically gone up (P < 0.05) compared to para-carcinoma tissues. The relative expression of LncRNA TDRG1 in cervical carcinoma was correlated with FIGO staging, lymph node metastasis, infiltrating depth of cervical basal, and the differentiation of cancer cells (P < 0.05). According to the results of the Kaplan-Meier curve and Log-rank test, the overall survival conditions of subjects with low-lncRNA TDRG1 were superior to that of those with high-lncRNA TDRG1 expression (P < 0.05). The expression of LncRNA TDRG1 in cervical carcinoma tissues and the clinicopathological features in predicting the overall survival (OS) in sufferers with cervical carcinoma were investigated by the Cox regression model. LncRNA TDRG1 expression in cervical carcinoma tissues is tightly associated with the progression and prognosis of cervical carcinoma, which may be a latent biological indicator for clinical diagnosis and prognosis of cervical carcinoma.

Relationship Between Expression of microRNA and Chemotherapy Resistance in Cervical Carcinoma

Cervical cancer (CC), although being a potentially avoidable disease, is the second most often diagnose gynecological cancer, with at minimum 530,000 new instant reported each year, and optimism for CC remains poor. Nearly half of individuals with locally advanced cervical cancer have a poor pathological response to standard therapy. As a result, research into the molecular pathogenesis of cervical cancer and associated therapeutic targets is a must. MicroRNAs (miRNAs) are possible biomarkers in cervical cancer; elevations or reductions in many distinct miRNAs discovered in individuals with this illness indicate that miRNA could contain a function to play in the illness's pathogenesis. Nevertheless, little is known about their significance in detecting individuals who do not respond to traditional therapy. As a consequence, the intention of this study is to look at the relationship among the synthesis of miRNAs (miR 217 and miR-140-3p), which can be utilized as molecular biomarkers to predict pathological responses in cervical cancer patients after radiation and chemotherapy. Various analytical techniques were used to analyze the data, including quantitative real-time PCR (qRT-PCR), growth and apoptosis analysis, western blot analysis, luciferase reporter gene analysis, immunohistochemistry, and statistical analysis. The results show that such miRNAs participate a crucial responsibility in CC cell proliferation inhibition. They might be a new therapeutic target for microRNA-mediated cell proliferation inhibition in cervical cancer.

Targeted Delivery of SmacN7 Peptide Induces Immunogenic Cell Death in Cervical Cancer Treatment

Cervical cancer is a common tumor in women and one of the common causes of cancer death in women. Due to the aggressive and non-specific nature of traditional chemotherapy, there is a growing need for new treatment modalities. Currently, tumor immunotherapy is increasingly garnering attention as a disruptive treatment approach. Therefore, we constructed CCTP-SmacN7, a delivery system capable of releasing active molecules in the tumor microenvironment. CCTP-SmacN7 can not only inhibit tumor proliferation and migration, but also induce tumors to produce large amounts of reactive oxygen species. The production of reactive oxygen species can activate tumors to release or expose damage-associated molecular patterns, promote DC cell maturation, and ultimately activate T cells. Here, we present an innovative targeted treatment approach for cervical cancer. While inducing tumor immunogenic cell death, this program can also improve the tumor microenvironment and initiate the tumor immune cycle.

Identification of Novel Genes and Pathways of Ovarian Cancer Using a Comprehensive Bioinformatic Framework

Ovarian cancer (OC) is a significant contributor to gynecological cancer-related deaths worldwide, with a high mortality rate. Despite several advances in understanding the pathogenesis of OC, the molecular mechanisms underlying its development and prognosis remain poorly understood. Therefore, the current research study aimed to identify hub genes involved in the pathogenesis of OC that could serve as selective diagnostic and therapeutic targets. To achieve this, the dataset GEO2R was used to retrieve differentially expressed genes. The study identified a total of five genes (CDKN1A, DKK1, CYP1B1, NTS, and GDF15) that were differentially expressed in OC. Subsequently, a network analysis was performed using the STRING database, followed by the construction of a network using Cytoscape. The network analyzer tool in Cytoscape predicted 276 upregulated and 269 downregulated genes. Furthermore, KEGG analysis was conducted to identify different pathways related to OC. Subsequently, survival analysis was performed to validate gene expression alterations and predict hub genes, using a p-value of 0.05 as a threshold. Four genes (CDKN1A, DKK1, CYP1B1, and NTS) were predicted as significant hub genes, while one gene (GDF15) was predicted as non-significant. The adjusted P values of said predicted genes are 2.85E - 07, 5.49E - 06, 4.28E - 07, 1.43E - 07, and 3.70E - 07 for CDKN1A, DKK1, NTS, GDF15, and CYP1B1 respectively; additionally 6.08, 5.76, 5.74, 5.01, and 4.9 LogFc values of the said genes were predicted in GEO data set. In a boxplot analysis, the expression of these genes was analyzed in normal and tumor cells. The study found that three genes were highly expressed in tumor cells, while two genes (CDKN1A and DKK1) were more elevated in normal cells. According to the boxplot analysis for CDKN1A, 50% of tumor cells ranged between approx 3.8 and 5, while 50% of normal cells ranged between approx 6.9 and 7.9, which is greater than tumor cells. This shows that in normal cells, the CYP1B1 has a high expression level according to the GEPIA boxplot; addtionally the boxplot for DKK1 indicated that 50% of tumor cells ranged between approx 0 and 0.5, which was less than that of normal cells which ranged between approx 0.3 and 0.9. It shows that DKK1 is highly expressed in normal genes. Overall, the current study provides novel insights into the molecular mechanisms underlying OC. The identified hub genes and drug candidate targets could potentially serve as alternative diagnostic and therapeutic options for OC patients. Further research is needed to investigate the clinical significance of these findings and develop effective interventions that can improve the prognosis of patients with OC.

Loureirin B inhibits Cervical Cancer Development by Blocking PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation

Loureirin B (LB) is an iconic component of Chinese dragon's blood that presents anti-cancer effects in gastric cancer and liver cancer. Although LB has shown benefits in treating several disorders such as cardiac fibrosis, cerebral ischemia/reperfusion, and osteoporosis, its effect on cervical cancer remains unknown. This study aimed to investigate the effects and mechanisms of LB on treating cervical cancer. A CCK-8 assay was conducted to determine the influence of LB on the viability of HeLa cells. Colony formation assay was performed to verify the impact of LB on HeLa cell proliferation. Cell cycle and apoptosis were detected by flow cytometry and western blot. The scratch assay, Transwell assay and western blot were used to examine the migration and invasion capacity of HeLa cells. The potential targets and signaling pathways of LB treating cervical cancer were predicted by network pharmacology analysis and subsequently validated in vitro. The results showed that the HeLa cell viability gradually declined to 64.83% for 12 h, 53.17% for 24 h, and 42.38% for 48 h after treatment with 5-80 μg/mL LB. Treatment with 20 μg/mL LB decreased cell colonies from 156.7 ± 11.7 to 102.7 ± 5.7. LB arrested cell cycle by reducing the expressions of Ki-67 and PCNA. Compared to the cell apoptosis rate of 2.63% in control group, LB increased it to 6.59% via upregulating Bax and suppressing Bcl-2 expressions. Additionally, LB reduced the invasion and migration capacity of HeLa cells by decreasing MMP-2 and MMP-9 levels. Network pharmacology analysis revealed that LB might suppress the PI3K/AKT signaling pathway to exert the aforementioned effects, as evidenced by a PI3K agonist attenuating the effects of LB on HeLa cells. In conclusion, this study demonstrated that LB inhibited the proliferation of cervical cancer cells, induced its apoptosis, and reduced its invasion and migration via targeting the PI3K/AKT signaling pathway.

Investigation of In Vitro Anti-cancer and Apoptotic Potential of Onion-Derived Nanovesicles Against Prostate and Cervical Cancer Cell Lines

Plant-derived compounds have recently garnered significant interest in the field of medicine due to their rich repertoire of phytochemicals, which holds promise for exploring novel therapies to treat cancer. This study embarks on the first-time investigation of the anti-cancerous effect of onion-derived nanovesicles (ODNVs). ODNVs were isolated employing differential centrifugation followed by ultracentrifugation and subsequent characterization using dynamic light scattering (DLS), field emission scanning electron microscopy (FESEM), and Fourier transform infrared spectroscopy (FTIR). Furthermore, we delineated the anti-cancerous effect of ODNVs on two cancer cell line models HeLa (cervical cancer) and PC-3 (prostate cancer) using MTT assay, DAPI-based DNA damage using immunofluorescence microscopy, colony formation assay, migration assay, cell cycle analysis, and evaluation of apoptosis using flow cytometry and western blotting. The findings revealed dose- and time-dependent anti-proliferative effects of ODNVs on both HeLa and PC3 cell lines, accompanied by selective cytotoxicity against cancer cells. Additional results highlighted that ODNVs prevented colony growth and induced S-phase cell cycle arrest. Apoptosis induction was evaluated through alterations in nuclear morphology and the number of apoptotic cells, which increased significantly after ODNV treatment in both cancer cell lines. Furthermore, annexin V/PI staining evaluation of apoptotic cells by flow cytometry demonstrated that ODNV treatment significantly increased the number of apoptotic cells in both PC-3 and HeLa cells. Finally, Western blot analysis indicated changes in apoptosis-related proteins including bcl-2, bax, and caspase-3, emphasizing that the anti-cancerous effect of ODNVs is attributed to the induction of apoptosis and suggests the  unexplored anti-cancerous potential of ODNVs.

Anticancer Effect of Active Component of Astragalus Membranaceus Combined with Olaparib on Ovarian Cancer Predicted by Network-Based Pharmacology

In China, a traditional Chinese medicine formulation called astragalus membranaceus (AM) has been utilised for more than 20 years to treat tumors with extraordinary effectiveness. The fundamental mechanisms, nevertheless, are still not well understood. The aim of this study is identifying its possible therapeutic targets and to evaluate the effects of AM in combination with a PARP inhibitor (olaparib) in the treatment of BRCA wild-type ovarian cancer. Significant genes were collected from Therapeutic Target Database and Database of Gene-Disease Associations. The components of AM were analyzed using the Traditional Chinese Medicine System Pharmacology (TCMSP) database to screen the active ingredients of AM based on their oral bioavailability and drug similarity index. In order to find intersection targets, Venn diagrams and STRING website diagrams were employed. STRING was also used to create a protein-protein interaction network. In order to create the ingredient-target network, Cytoscape 3.8.0 was used. DAVID database was utilized to carry out enrichment and pathway analyses. The binding ability of the active compounds of AM to the core targets of AM-OC was verified with molecular docking using AutoDock software. Experimental validations, including cell scratch, cell transwell, cloning experiment, were conducted to verify the effects of AM on OC cells. A total of 14 active ingredients of AM and 28 AM-OC-related targets were screened by network pharmacology analysis. The ten most significant Gene Ontology (GO) biological function analyses, as well as the 20 foremost Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were selected. Moreover, molecular docking results showed that bioactive compound (quercetin) demonstrated a good binding ability with tumor protein p53 (TP53), MYC, vascular endothelial growth factor A (VEGFA), phosphatase and tensin homolog (PTEN), AKT serine/threonine kinase 1 (AKT1) and cyclin D1 (CCND1) oncogenes. According to experimental methods, in vitro OC cell proliferation and migration appeared to be inhibited by quercetin, which also increased apoptosis. In addition, the combination with olaparib further enhanced the effect of quercetin on OC. Based on network pharmacology, molecular docking, and experimental validation, the combination of PARP inhibitor and quercetin enhanced the anti-proliferative activity in BRCA wild-type ovarian cancer cells, which supplies the theoretical groundwork for additional pharmacological investigation.

Silencing E6/E7 Oncoproteins in SiHa Cells Treated with siRNAs and Oroxylum indicum Extracts Induced Apoptosis by Upregulating p53/pRb Pathways

E6 and E7 human papillomavirus (HPV) oncoproteins play a significant role in the malignant transformation of infected cervical cancer cells via suppression of tumour suppressor pathways by targeting p53 and pRb, respectively. This study aimed to investigate the anticancer effects of Oroxylum indicum (OI) leaves' methanol extract on SiHa cervical cancer cells. Expression of apoptosis-related proteins (Bcl-2, caspase (cas)-3, and cas-9), viral oncoproteins (E6 and E7), and tumour suppressor proteins (p53 and pRb) were evaluated using western blot analysis before and after E6/E7 small interfering RNAs (siRNAs) transfection. In addition, the E6/E7 mRNA expression levels were assessed with real-time (RT)-PCR. The present study showed that the OI extract effectively hindered the proliferation of SiHa cells and instigated increments of cas-3 and cas-9 expressions but decreased the Bcl-2 expressions. The OI extract inhibited E6/E7 viral oncoproteins, leading to upregulation of p53 and pRb tumour suppressor genes in SiHa cells. Additionally, combinatorial treatment of OI extract and gossypin flavonoid induced restorations of p53 and pRb. Treatment with OI extract in siRNA-transfected cells also further suppressed E6/E7 expression levels and further upregulations of p53 and pRb proteins. In conclusion, OI extract treatment on siRNAs-transfected SiHa cells can additively and effectively block E6- and E7-dependent p53 and pRb degradations. All these data suggest that OI could be explored for its chemotherapeutic potential in cervical cancer cells with HPV-integrated genomes.

LncRNA LINC00665 Promotes Ovarian Cancer Cell Proliferation and Inhibits Apoptosis via Targeting miR-181a-5p/FHDC

Previous reports indicate that long intergenic non-coding RNA LINC00665 naturally occurred vital effects in various cancers. Herein, the role of LINC00665 in ovarian cancer progress was explored. We found that LINC00665 was upregulated in ovarian cancer cell lines. Besides, a series of assays including flow cytometry, wound-healing, transwell, cell counting Kit-8 (CCK-8), and EdU assay confirmed that the knockdown of LINC00665 could reduce the viability, proliferation, and migration of SKOV-3 and OVCAR-3 cells. Accumulating evidence indicates that many lncRNAs can function as endogenous miRNA sponges by competitively binding common miRNAs. In this study, the bioinformatics analysis suggests that LNC00665 specifically binds to miR-181a-5p. LINC00665 downregulated the miR-181a-5p in SKOV-3 and OVCAR-3 cells. The knockdown of miR-181a-5p evidently reverses the inhibitory effect of sh-LINC00662. Besides, FH2 domain containing 1 (FHDC1) has been proved to deed as an effective target of miR-181a-5p. The results reveal the knockdown of LINC00665 facilitates ovarian cancer via development by sponging miR-181a-5p and up-regulating FHDC1 expression. These may contribute to ovarian cancer therapy.

Inhibition of Proliferation in Ovarian Cancer Cell Line (PA-1) by the Action of Green Compound “Betanin”

Ovarian carcinoma has a cure rate of 30% which makes it deadlier than any other disease. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Chemoprevention of cancer through application of natural compounds is the need of present generation as other methods are rigorous and have many side effects. Betanin, a compound from Beta vulgaris extract is used in present study to check its potential for inhibition of (PA-1) cancer cell proliferation. Determination of IC50 values through MTT assay was carried out, in addition measurement of mitochondrial membrane potential (MMP), effect of reactive oxygen species (ROS) generation, and induction of apoptosis in ovarian cancer cells through betanin was also observed. Results have shown betanin as a potential candidate for inhibition of ovarian cancer cell proliferation and it can be taken up as a serious compound for further studies for its application in cancer cure.

EXT1 and Its Methylation Involved in the Progression of Uterine Corpus Endometrial Carcinoma Pathogenesis

Uterine corpus endometrial carcinoma (UCEC) is one of the most common gynecologic tumors. Due to the high recurrence and metastasis of UCEC, it is crucial for patients to find new biomarkers for diagnosis and therapy. In this study, R software and the TCGA database were used to screen candidate UCEC predictive markers. Western blot and RT-qPCR were performed to detect protein and mRNA expression of EXT1 in UCEC cell lines. In addition, MTT assay, flow cytometry, transwell assay, and wound healing assay were conducted to assess the cell viability, apoptosis, invasion, and migration in UCEC cells. Overlap-extension PCR technique was employed to construct the vector targeting the deletion of the methylated segment of EXT1. The results showed that a total of 11 candidate genes were obtained and EXT1 was identified as a potential target. The expression and methylation levels of EXT1 were both increased in UCEC tissues and cell lines, as well as elevated EXT1 was closely related to the poor prognosis of patients. Besides, the knockdown of EXT1 significantly inhibited the malignant biological behaviors in UCEC cells. Additionally, the current study also found that the deletion of 1559-2146 bp CpG island segment upregulated EXT1 expression and promoted malignant biological behaviors in UCEC cells. Furthermore, the presence of m7G RNA methylation in UCEC cells also was found. In conclusion, the methylation of EXT1 influenced the gene expression, thereby affecting the malignant biological behaviors in UCEC cells and regulating the pathological progression of UCEC.

lncRNA ENST00000585827 Contributes to the Progression of Endometrial Carcinoma via Regulating miR-424/E2F6/E2F7 Axis

Endometrial cancer (EC) ranks fourth among the most common gynecologic malignancies. Despite advances in medical technology, the pathogenesis is still unclear. Numerous reports have identified the involvement of lncRNA in the malignant progression of endometrial cancer. The aim of the study was to investigate the expression level of lncRNA ENST00000585827 (lncRNA E27) in endometrial cancer and the molecular mechanism that regulates the development of endometrial cancer. Combined with the results of the previous study, PCR analysis confirmed that lncRNA E27 was significantly upregulated in endometrial cancer cell lines. The results of CCK-8, wound healing assay, and transwell experiments showed that lncRNA E27 could significantly inhibit cell proliferation, migration, and invasion. Flow cytometry results confirmed that lncRNA E27 could promote apoptosis. Furthermore, based on bioinformatics predictions, dual-luciferase assay and RT-qPCR analysis confirmed that miR-424, as its downstream molecule, competitively regulates the expression of E2F6/E2F7. Rescue experiments further supported that lncRNA E27 inhibited proliferation, migration, invasion, and promoted apoptosis of endometrial cancer through miR-424/E2F6/E2F7 signaling axis. Conclusively, our findings revealed the role of lncRNA E27 in regulating the miR-424/E2F6/E2F7 signaling axis during EC progression, opening up new strategies for the treatment of endometrial cancer.