Dual Staining Research Articles

In vitro cytotoxicity and apoptosis inducing effect of Vallarai Kirutham with Rasa Parpam in HeLa Cell Lines

Cancer is an uncontrolled proliferation of cells that can affect nearby tissues as well as distant organs. Cervical cancer is predicted to be among the four most prevalent cancers in women overall in 2020, with 342,000 mortality and 604,000 new cases. Around 6-29% of all cancers in women in India are caused by cervical cancer. Women who have had several sexual partners, recurrent abortions, or vaginal deliveries—all of which cause repetitive stress to the cervix are more likely to develop cervical cancer. Radiation, chemotherapy, and surgery are being used to treat cancer. All of these treatments come with side effects, which can range in severity and have a serious psychological impact on patients. The Siddha system has a number of accessible herbo mineral formulations for treating cervical cancer. According to Siddha literature (Agasthiyar Vaidhya Rathina Churukkam, 360), the treatment of cervical cancer (yoni puttru) is advised for the Siddha medicine Vallarai Kirutham with Rasa parpam. The goal of this research was to find out this formulation's potential anti-cancer properties using MTT assays on HeLa cell lines. These findings of the current study suggest that the MTT technique's lowest reading for cell viability was 1.26±0.009% at a concentration of 100 µl/ml. This was followed by concentrations of 75 µl/ml, 50µl/ml and 25µl/ml which showed 7.91±0.004%, 23.91 ±0.01% and 37.17± 0.01% similarly, 10 µl/ml shows 52.09±0.01%.The matching IC50 value was discovered to be 12.44% and AO/EB dual staining was used to examine apoptotic activity. The outcomes of in-vitro experiments using the HeLa cell line. According to the outcome of in vitro experiments done on the HeLa cell line, the drug had substantial anti-cancer and therapeutic value in the management of cervical tumours .With a variety of time tested medications the Siddha system has shown outcomes that were clinically important for the management of cervical carcinoma. One of those medications has to undergo additional testing in patients with cervical cancer through clinical research.

In vitro combination effects of plant-derived quercetin with synthetic bicalutamide on prostate cancer and normal cell lines: in silico comparison.

Prostate cancer is the second most frequent and the fifth greatest cause of death in men. Although diet has been connected to the prevalence of cancer in addition to other factors, the relation between cancer and prevention is weak. Treatment options are at risk due to cell resistance. To identify new combinations, we tried plant-derived quercetin with bicalutamide on cell lines. To determine the cytotoxicity and apoptotic potential of plant-derived quercetin and its combination, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] and dual stain assays were performed. In silico protein-ligand interaction was performed to support the in vitro findings. A thin layer, column, and high-performance chromatography were used to purify quercetin along with an authentic sample. In the cytotoxic study, quercetin was minimized by 80% similar to bicalutamide and a combination of quercetin and bicalutamide by 50% when compared to controls by 2%. Quercetin and bicalutamide showed a similar binding affinity for androgen receptors (9.7 and 9.8), hub genes (10.8 and 10.0), and a few other PCa-related genes (9.4 and 9.1). We propose to conclude that the combination of quercetin plus bicalutamide can be used for chemotherapy if additional in vivo studies are conducted. The intake of foods high in polyphenolic compounds can help to prevent prostate cancer. Examination of quercetin on several cell lines will provide a definite conclusion to combat cancers.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2

BackgroundPremature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear.MethodsNinety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.ResultsBy WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.ConclusionOur findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Abstract 5930: Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method

Abstract Ovarian cancer accounts for approximately 6% of all cancer death in women and has one of the highest mortality rates out of all gynecologic malignancies in the United States. Ovarian cancer can exhibit innate or acquired chemoresistance behavior, thus it is critical to identify novel therapeutic drug candidates. One of important characteristics of identifying potential chemotherapy drug candidate is the ability to induce apoptosis or programmed cell death in the target cancer cells. In order to measure apoptosis, various biomarkers are commonly employed such as Caspase 3, 7, 8 or 9, as well as Annexin V, which are all part of the cascade in apoptotic intrinsic pathway. However, typical fluorescent staining for image cytometry is not multiplexed that required multiple steps in order to determine the different type of apoptotic populations. In this work, we demonstrate a multiplexing apoptosis detection method to investigate the apoptotic effects of EP4A1, EP4A2, Paclitaxel, and Carboplatin on Kuramochi ovarian cancer cells. We employed the use of a high-throughput plate-based image cytometer (Celigo, Revvity Health Sciences Inc.) to image and analyze drug-treated Kuramochi cells stained with Annexin V-APC, Caspase 3/7 (488), and propidium iodide (PI) to assess the early- and late-stage apoptosis, as well as necrosis. We expected to see an increase in dual staining with the addition of EP4A to paclitaxel and carboplatin; however, the more interesting data was observed by separating single positive cell populations (Caspase, Annexin or PI only) from dual positive cell populations (Annexin + PI, Caspase + PI, Annexin +PI). The results show that treatment with EP4A lead to a time dependent increase in Caspase 3 cleavage but only in the single positive cell population. This trend was also observed in Annexin V single positive cell populations which is an indication of early apoptosis. The additive effect observed with the addition of EP4A was not seen in the dual positive cell populations. The high throughput nature of the image cytometry also allowed us to easily and quickly determine the optimal treatment conditions for this experiment. We were able to record multiple time points and not only capture proliferation, but also simultaneously collect data on caspase cleavage, Annexin-V staining and PI staining on multiple concentrations of compounds in monotherapy, dual therapy, and triple therapy combinations. Typically, it may be difficult to test multiple conditions in duplicate and triplicate using flow cytometry, while Celigo Image Cytometry may be used to obtain more robust data, and simultaneously acquire data of proliferation and multiplex staining with Annexin-V, PI and Caspase 3/7. Citation Format: James McDonald, Leo Li-Ying Chan, Jocelyn Reader, Sergio Mojica, Mackenzie Pierce. Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5930.

Abstract 7307: Silibinin regulates glucocorticoid signaling via mast cells: Potential treatment of androgen-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men. The American Cancer Society estimates that in 2023 there have been ~288,300 new cases of PCa and ~34,700 deaths from PCa in the United States. We previously showed that silibinin (SB; a natural flavonolignan from milk thistle seeds) is an effective therapeutic against PCa that inhibits cell growth and mitogenic cell survival via EGFR, IGFR-1, and NFκB signaling. However, the effect of SB on the tumor immunological microenvironment has not been elucidated. Mast cells (MCs) are archetypal solider of the immune system. The dynamics of PCa and MCs are complex, where they may contribute to tumor transformation/progression/regression; furthermore, their role in PCa is poorly understood. To address this gap and understand the role of SB in MC regulation, MCs were derived from the bone marrow (BMMCs) of C57BL/6 mice. BMMCs were generated in the presence of IL-3 and SCF, and were confirmed to be BMMCs using flow cytometry (dual staining: cKit/Fc€RI). Thereafter, the BMMCs were exposed to different doses of SB (25-100 µM) and two concentrations (25 µM; SB 25 and 100 µM; SB 100) were selected for further experiments. BMMCs treated with SB 25, SB 100, and untreated (control) were subjected to proteomic analysis using LCMS on Fusion Lumos mass spectrometer. A total of 3575 proteins were identified using the annotated mouse proteome. Statistical analysis was performed using ANOVA followed by Fisher’s posthoc analysis (p<0.05, FDR<0.01) and 166 proteins were found to be have statistically significant differential expression amongst the three groups (n=4/group). These 166 proteins were selected for further pathway analysis using Ingenuity Pathway Analysis (IPA; Qiagen). Only pathways specifically demonstrated experimentally in prostatic tissues/cells were selected for further analysis. Important nodes found for SB regulation of MCs in PCa were androgen receptor (AR), transmembrane serine protease (TMPRSS), and NK3 Homeobox 1 (NKX3-1). AR, TMPRSS, and NkX3-1 are well established in PCA. Most important pathway found was glucocorticoid receptor (GR) signaling; nuclear receptor subfamily 3 group C member 1 (NR3C1) and HS90 were the most important proteins identified in GR signaling. GR signaling may cause androgen deprivation therapy (ADT)-resistance by directly activating AR-target genes (including TMPRSS2) and/or activating an independent transcriptome. NR3C1 has been shown to share target specificity with AR and is highly upregulated in the resistant tumors. Thus, SB could play an important therapeutic role, specifically in ADT-resistant tumors via regulation of MCs. Further studies are warranted to delineate these mechanisms. Understanding these mechanistic could also potentially aid to decipher a general mechanism of resistance to antiandrogens and potential of SB in treating ADT-resistant tumors. Citation Format: Neha Mishra, Sandeep Paudel, Komal Raina, Paul Maroni, Chapla Agarwal, Rajesh Agarwal. Silibinin regulates glucocorticoid signaling via mast cells: Potential treatment of androgen-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7307.

Abstract 6797: CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application

Abstract Lung cancer (LC) is the most prevalent type of cancer globally and the leading cause of cancer-related deaths. Based on the histology and type of treatments, LC is divided into small-cells and non-small cell lung cancer (NSCLC) carcinomas, which are more aggressive and less responsive to chemotherapy. Since LC is known to be a highly heterogeneous cell population, treatment is challenging. It has been observed that chemokine receptor-ligand interaction is essential for cancer cells homing to distant sites. Most malignancies, including LC, have established disease status indicators concerning elevated levels of CCL4, a structurally unique chemokine, and its receptor (CCR5). Therefore, treatment strategies that inhibit the LC cell's tumor environment may improve patients' quality of life. However, the CCR5/CCL4 interface in LC invasion and metastasis is poorly understood. In this study, we examined the expression and function of the CCR5/CCL4 axis in the development and invasiveness of LC patient's clinical tissues and cell lines. We conducted immunohistochemistry (IHC) dual staining of CCR5/CCL4 antibodies in LC tissues. Our findings demonstrated significantly (p < 0.001) higher expression of CCR5 and CCL4 in advanced-stage malignant tissues than in normal tissues. The in-vitro analysis of the human LC cell lines NCI-H1437, NCI-H1573, and NCI-H1975 revealed CCL4 activation expressing higher CCR5, determined by immunofluorescence, western blots, flow cytometry, 3D-invasion assay, and RT-qPCR methods. Further, a calcium mobilization assay confirmed the direct interaction between CCR5 and CCL4. However, treating with Maraviroc, a small molecule inhibitor of CCR5, to LC cells blocks their ability to proliferate, demonstrating the crucial role that CCR5-CCL4 chemotaxis plays in LC metastasis. Our findings show both the biological and clinical significance of CCR5 and CCL4 expression, which may serve as potential indicators for metastatic LC. Therefore, inhibiting the CCR5/CCL4 interaction may offer a more effective therapeutic approach for managing the progression of LC. Citation Format: Santosh K. Singh, Rajesh Singh. CCR5/CCL4 axis in lung cancer clinicopathological significance and its therapeutic application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6797.

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.

Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid Production

Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.

Recommendations for Use of p16/Ki67 Dual Stain for Management of Individuals Testing Positive for Human Papillomavirus.

The Enduring Consensus Cervical Cancer Screening and Management Guidelines Committee developed recommendations for dual stain (DS) testing with CINtec PLUS Cytology for use of DS to triage high-risk human papillomavirus (HPV)-positive results. Risks of cervical intraepithelial neoplasia grade 3 or worse were calculated according to DS results among individuals testing HPV-positive using data from the Kaiser Permanente Northern California cohort and the STudying Risk to Improve DisparitiES study in Mississippi. Management recommendations were based on clinical action thresholds developed for the 2019 American Society for Colposcopy and Cervical Pathology Risk-Based Management Consensus Guidelines. Resource usage metrics were calculated to support decision-making. Risk estimates in relation to clinical action thresholds were reviewed and used as the basis for draft recommendations. After an open comment period, recommendations were finalized and ratified through a vote by the Consensus Stakeholder Group. For triage of positive HPV results from screening with primary HPV testing (with or without genotyping) or with cytology cotesting, colposcopy is recommended for individuals testing DS-positive. One-year follow-up with HPV-based testing is recommended for individuals testing DS-negative, except for HPV16- and HPV18-positive results, or high-grade cytology in cotesting, where immediate colposcopy referral is recommended. Risk estimates were similar between the Kaiser Permanente Northern California and STudying Risk to Improve DisparitiES populations. In general, resource usage metrics suggest that compared with cytology, DS requires fewer colposcopies and detects cervical intraepithelial neoplasia grade 3 or worse earlier. Dual stain testing with CINtec PLUS Cytology is acceptable for triage of HPV-positive test results. Risk estimates are portable across different populations.

Synthesis of Zinc Oxide Nanoflakes by Non-Thermal Plasma Technique and Evaluation of Anticancer Activity in Vitro

This paper describes the synthesis of ZnO nanoparticles using air microplasma jets as the main reaction medium. Various techniques, including X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and UV—Vis spectroscopy, were employed to confirm the characterization of the artificially synthesized ZnO nanoparticles. The absence of additional peaks associated with secondary phases of ZnO, as revealed by the XRD analysis, indicates the exceptional purity of these nanoparticles. Through the use of SEM, we examined the surface morphology and observed a substantial rate of agglomeration. The energy band gaps of ZnO nanoparticles were determined to be 3.3 electron volts (eV). The cytotoxic effects of zinc oxide nanoparticles were assessed using the methyl thiazolyl tetrazolium (MTT) assay on breast cancer cells exposed to various doses of the nanoparticles. The Acridine-Orange/Ethidium-bromide (AO/EB) dual stain was employed to assess apoptotic cells. The data indicate that ZnO NPs can induce apoptosis, suggesting their potential as an anticancer treatment in breast cancer cell lines. The presented findings indicate that nanoscale zinc oxide particles (ZnO NPs) possess significant potential for future utilization in many biological applications. An example of such an application could be the prospective substitution for chemotherapy in the management of various forms of cancer diseases.

Should we use risk selection tests for HPV 16 and/or 18 positive cases: Comparison of p16/Ki67 and cytology.

Major screening abnormalities in precolposcopic stage are tests results that imply direct referral to colposcopy (and/or expedited treatment) without performing additional high-grade squamous intraepithelial lesions or worse (HSIL+) risk selection testing. Currently, both clinically validated HSIL+ risk selection tests, reflex cytology and reflex p16/Ki67 dual staining (DS), are being compared for use in primaryhuman papillomavirus (HPV)-based screening to avoid possible overtreatment, but there is still no sufficient data available for their performance. Among 30 066 liquid-based cervical cancer screening tests results, a group of 332 women was selected with available high-risk types ofHPV tests results with 16/18 limited genotyping, liquid-based cytology, DS, and histology results from standardized colposcopy with biopsy. In HPV 16/18+ cases, three triage approaches were retrospectively analyzed. Predictive values for detection of HSIL+ were calculated and number of colposcopies required in each strategy. Both triage models with DS used (reflex cytology followed by DS, and reflex DS alone in all cases) had significantly higher positive predictive valuefor HSIL+ than strategy with reflex cytology alone (44.2%/45.7% vs. 28.3%; p < 0.0001). In models with DS, less colposcopies were required (95/92 vs. 152) and less colposcopies were needed per HSIL+ detection (2.26/2.19 vs. 3.54). Only oneHSIL+ case was missed in both triage models with DS incorporation. p16/Ki67 dual-stain may be an effective, alone or combined with cytology, triage test to detect HSIL+ in patients with major screening abnormalities in primary HPV-based cervical cancer screening. Performing cytology as the first triage test improves the strategy by enabling referrals to expedited treatment in selected cases.

Biogenic Zinc Oxide Nanoparticles Attenuate Acute Lymphoblastic Leukemia Cell Proliferation through Oxidative Stress and DNA Damage

Background: The most prevalent pediatric cancer is acute lymphoblastic leukemia (ALL). It is exceedingly challenging to treat recurrent diseases, and there aren’t many new medications available for children with disease resistance. The size-dependent anticancer effect of zinc oxide nanoparticles (ZnONPs) on T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT 3 is the central theme of this study. Methods: The leaf and bark extracts of Diospyros montana were subjected to nanoparticle (NPs) synthesis and characterized analytically to acquire ZnONPs. The ZnONPs were characterized using UV-Vis spectra, DLS, XRD, EDX, SEM, and TEM analysis. Then the ZnONPs were separated into two groups having &lt;50 nm and 50-100 nm NPs sizes. In addition, MTT assay, dual staining, autophagy, DNA damage, and oxidative damage measurement were done to find out the anti-cancer effect of the ZnONPs on MOLT 3 cells. Results: The ZnONPs exhibited a size-dependent anticancer effect against MOLT 3 cells. The IC50 of ZnONPs of &lt; 50 nm was found to be ~75 µg/mL while the IC50 of ZnONPs of 50-100 nm size was found to be ~102 µg/mL. Treatment with ZnONPs of &lt;50 nm size decreased mitochondrial membrane potential, more than that with 50-100 nm ZnONPs. On the other hand, autophagy was found to be more prevalent in 50-100 nm treated ZnONPs when compared with &lt; 50 nm size. However, treatment with both sizes of ZnONPs reduced cell proliferation markers such as ki67 positive cells while increasing 8-OHG and HDCF-DA positive cells. Conclusion: The obtained results portrayed that different-sized ZnONPs induce different modes of T-ALL cell death. Small-sized ZnONPs revealed higher efficacy, highlighting the size-dependent property. Further, this finding denotes that ZnONPs could be an effective anticancer agent against dreadful diseases like T-ALL, warranting further investigation.

A Prospective Study on the Progression, Recurrence, and Regression of Cervical Lesions: Assessing Various Screening Approaches.

(1) Background: The prediction of cervical lesion evolution is a challenge for clinicians. This prospective study aimed to determine and compare the predictive accuracy of cytology, HPV genotyping, and p16/Ki67 dual staining alone or in combination with personal risk factors in the prediction of progression, regression, or persistence of cervical lesions in human papillomavirus (HPV)-infected patients; (2) Methods: This prospective study included HPV-positive patients with or without cervical lesions who underwent follow-up in a private clinic. We calculated the predictive performance of individual tests (cervical cytology, HPV genotyping, CINtecPlus results, and clinical risk factors) or their combination in the prediction of cervical lesion progression, regression, and persistence; (3) Results: The highest predictive performance for the progression of cervical lesions was achieved by a model comprising a Pap smear suggestive of high-grade squamous intraepithelial lesion (HSIL), the presence of 16/18 HPV strains, a positive p16/Ki67 dual staining result along with the presence of at least three clinical risk factors, which had a sensitivity (Se) of 74.42%, a specificity of 97.92%, an area under the receiver operating curve (AUC) of 0.961, and an accuracy of 90.65%. The prediction of cervical lesion regression or persistence was modest when using individual or combined tests; (4) Conclusions: Multiple testing or new biomarkers should be used to improve HPV-positive patient surveillance, especially for cervical lesion regression or persistence prediction.

Copper(II) complexes incorporated with 2,2-bipyridyl and 2,2′-(((1E,1′E)-1,4-phenylene-bis(methanylylidene))bis(azanylylidene)) diphenol derivatives: In-vitro interaction with DNA/BSA, DFT, molecular docking and cytotoxicity

Certain metal complexes have created their own benchmark in the category of cytotoxic drugs. Down the line, copper metal complexes are found among the most prominent biologically active agents of high reputation. Considering breast cancer as one of the epicenters in malignant tumors, a series of three new derivatives of copper(II) CuL1, CuL2 and CuL3 have been synthesized and characterized associated with newly developed ligands 2,2′-(((1E,1′E)-1,4-phenylene-bis(methanylylidene))bis(azanylylidene)) diphenol supported by 2,2-bipyridine. All the complexes appeared in tetracoordination with tetrahedral geometry. The complexes were made to bind with ct-DNA through groove binding as established by molecular docking and computational DFT studies carried out for the determination of the HOMO and LUMO interactions and ESP mapping. Further, confirmation of BSA binding with complexes was confirmed by UV and fluorescence spectral studies. All these copper complexes were subjected for DPPH assay to determine the antioxidant properties, cytotoxicity towards the breast adenocarcinoma was retrieved by MTT assay, dual staining and cell cycle analysis against MCF7 cell line. Eventually, new copper derivative CuL2 (IC50 = 30.20 μg/mL) was found to exhibit the highest anticancer activity as compared to CuL1 (IC50 = 41.07 μg/mL) and CuL3 (IC50 = 42.01 μg/mL).

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 24h 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 IC50 of 21.6 µM after 24-h incubation. Additionally, HeLa cells treated with FN and SFN together showed increased apoptotic signals and considerable ROS generation. Consequently, by preventing the production of PI3K, AKT, and mToR-mediated regulation of proliferation and cell cycle-regulating proteins, the combined use of FN + SFN has been regarded as a chemotherapeutic medication. Further research will need to be done shortly to determine how effectively the co-treatment promotes apoptosis to employ them economically.

Enhancing Cervical Cancer Screening: Review of p16/Ki-67 Dual Staining as a Promising Triage Strategy.

Cervical cancer, primarily caused by high-risk human papillomavirus (HR-HPV) types 16 and 18, is a major global health concern. Persistent HR-HPV infection can progress from reversible precancerous lesions to invasive cervical cancer, which is driven by the oncogenic activity of human papillomavirus (HPV) genes, particularly E6 and E7. Traditional screening methods, including cytology and HPV testing, have limited sensitivity and specificity. This review explores the application of p16/Ki-67 dual-staining cytology for cervical cancer screening. This advanced immunocytochemical method allows for simultaneously detecting p16 and Ki-67 proteins within cervical epithelial cells, offering a more specific approach for triaging HPV-positive women. Dual staining and traditional methods are compared, demonstrating their high sensitivity and negative predictive value but low specificity. The increased sensitivity of dual staining results in higher detection rates of CIN2+ lesions, which is crucial for preventing cervical cancer progression. However, its low specificity may lead to increased false-positive results and unnecessary biopsies. The implications of integrating dual staining into contemporary screening strategies, particularly considering the evolving landscape of HPV vaccination and changes in HPV genotype prevalence, are also discussed. New guidelines and further research are necessary to elucidate the long-term effects of integrating dual staining into screening protocols.

Anticancer effects of rutin from Fagopyrum tataricum (tartary buckwheat) against osteosarcoma cell line.

The present study is analysisof the seeds of buckwheat (Fagopyrum sp.),member of the Polygonaceae family for isolation of rutin and its anticancer property againstOsteosarcoma celllines (SAOS2). The selected plant is traditionally used for diabetes and cancer. It has several biological properties such as antibacterial, antioxidant and anti-aging. Thirty-five buckwheat cultivars were obtained from Nepal Agriculture Genetic Resources Centre (NAGRC) Khumaltar, Kathmandu, Nepal, andKumrek Sikkim. These plant varieties are scientifically evaluated their biological properties. Rutin wasfractionated from buckwheat seeds using methanol fraction and analysed for quality by HPLC method. The rutin fraction of the cultivar NGRC03731 a tartary buck wheat and standard rutin was used against Osteosarcoma cell lines (SAOS2) and human gingival fibroblast cells (hGFs) for anticancer activity. The cell viability using rutin fraction and standard rutin treated with SAOS2 cells were assessed by MTT assay. For further research, the best doses (IC-50: 20g/ml) were applied. By using AO/EtBr dual staining, the effects of Rutin fraction on SAOS2 cell death were analysed. The scratch wound healing assay was used to analyse cell migration. Real-time PCR was used to analyse the pro-/anti-apoptotic gene expression. The seeds with the highest rutin content, NGRC03731 seeds, had 433mg/100g of rutin.The rutin fraction treatment and standard rutin significantly reduced cell viability in the MTT assay, and osteosarcoma cells were observed on sensitive to the IC-50 dose at a concentration of 20g/ml after 24h.The SAOS2 cells exposed to rutin fraction at 20g/ml and standard rutin at 10g/ml exhibited significant morphological alterations, cell shrinkage and decreased cell density, which indicate apoptotic cells.Rutin-fraction treated cells stained with acridine orange/ethidium bromide (AO/EtBr) dual staining cells turned yellow, orange, and red which indicatesto measure apoptosis.The anti-migration potential of rutin fraction, results prevented the migration of SAOS2 cancer cells.Rutin-fraction significantly increased the expression of pro-apoptotic proteinsBad, using real-time PCR analysis (mRNA for Bcl-2 family proteins) resulted Bcl-2's expression is negatively regulated. Osteosarcoma (SAOS2) cell lines' proliferation, migration, and ability to proliferate were reduced markedly by rutin fraction and it also causes apoptosis of Osteosarcoma cell lines (SAOS2).

Morin Inhibits Cell Proliferation and Induces Caspase-mediated Apoptotic Cell Death in Glioma C6 Cell Line

Background Glioma is a recurrent form of primary malignant cancer that occurs in the brain and central nervous system. The adults are the major victims of gliomas, and the men are mostly affected. At present, gliomas are treated with surgical resection followed by radiation and the administration of chemodrugs such as temozolomide. The invasive and high recurrence rate of gliomas often reduces the effectiveness of treatment, which leads to poor recovery. Hence, more potent drugs without side effects on long-term treatment need to be discovered to treat gliomas. Morin is one such promising phytochemical that possesses immense pharmacological properties. It is proven to exert anti-inflammatory, antioxidant, anticancer, antibacterial, antidiabetic, and neuroprotective properties. The current research focuses on examining the growth inhibition and apoptosis-inducing potency of morin against glioma C6 cells. Materials and Methods Rat C6 glioma cells were treated with different concentrations of morin and analyzed for cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The intracellular reactive oxygen species (ROS) production and alteration of mitochondrial membrane potential (MMP) by morin in glioma cells were examined with dichlorodihydrofluorescein diacetate (DCHF-DA) and rhodamine 123 staining. The apoptotic induction in glioma cells was analyzed with dual staining and it was confirmed by quantifying the apoptotic protein using the enzyme-linked immunosorbent assay (ELISA) technique. The anti-inflammatory property of morin was assessed by the inflammatory cytokines tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) using the ELISA technique. Results Our MTT results show that morin treatment significantly reduced the cell viability of rat C6 glioma cells. It also significantly increased ROS generation and decreased MMP in rat C6 glioma cells. Morin treatment also effectively increased caspase-3 and caspase-9, the proapoptotic protein Bax, and decreased the antiapoptotic protein Bcl-2. The induction of apoptosis by morin in glioma cells was evident with our acridine orange/ethidium bromide (AO/EtBr) staining results. Morin also decreased the inflammatory cytokine levels in rat C6 glioma cells. Conclusion Our findings have proven that morin significantly induces apoptosis by increasing the levels of apoptotic protein via the generation of ROS. It also effectively reduced inflammatory cytokine levels, thereby exerting anticancer activity against glioma cells. Therefore, morin can be subjected to further research to be designed as a potent anticancer drug to treat gliomas.

Doxorubicin-induced chemoresistance in Duke's type B colon adenocarcinoma cell line is aggravated in the presence of TGF-β2 through non-apoptotic cell death.

The current challenge in clinical cancer treatment is chemoresistance. Colon cells have inherently higher xenobiotic transporters expression and hence can attain resistance rapidly. Increased levels of TGF-β2 expression in patients have been attributed to cancer progression, aggressiveness, and resistance. To investigate resistance progression, we treated doxorubicin (dox) to HT-29 colon adenocarcinoma cells in the presence or absence of TGF-β2 ligand. After 1, 3-, and 7-day treatment, we investigated cell proliferation, viability, and cytotoxicity by MTT, trypan blue staining, and lactate dehydrogenase enzyme release. The mechanism of cell death was elucidated by hoechst33342 and propidium iodide dual staining and apoptosis assay. The development of resistance was detected by rhodamine123 efflux and P-glycoprotein (P-gp)/MDR1 antibody staining through fluorimetry and flow cytometry. The colony formation ability of the cells was also elucidated. Inhibition of cell proliferation was noted after day 1, while a significant reduction in viability and a significant increase in lactate dehydrogenase release was detected after day 3. Reduction of intracellular rhodamine123 levels was detected after day 3 and was significantly lower in dox with TGF-β2 treatment compared to dox alone. Increased surface P-gp levels after days 3 and 7 were observed in the treated groups. Hoechst33342/propidium iodide staining and apoptosis assay indicated non-apoptotic cell death. The cells treated with TGF-β2 had higher colony formation ability. TGF-β2 expression might play a significant role in the development of chemoresistance to doxorubicin in Duke's type B colon adenocarcinoma cell line, HT-29.

Abstract B016: Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining

Abstract Using gene expression profiling data, we previously identified three immune subtypes (low-TIL, high-TIL, high-ISG) of breast cancer (BC) among Chinese BC patients in Hong Kong (HK) (Zhu et al., Breast Cancer Res, 2017). To investigate if standard in situ immunohistochemical (IHC) staining could capture the BC immune landscape, we analyzed archival tissue sections from 148 HKBC patients who were previously characterized based on gene expression. Dual IHC staining for 8 immune markers (CD3, CD8, CD20, FOXP3, CD68, CD163, PDL1, and IDO1) and PanCK was performed. Digitized whole slide images for each marker were analyzed using high-accuracy machine learning algorithms trained to quantify the percentage of positively stained regions. Strong correlations were found between IHC immune markers and gene expression-based immune and intrinsic subtypes. Compared to patients with lower level of tumor-infiltrating lymphocytes (low-TIL) classified based on gene expression data, those in the high-TIL subtype (characterized by high T cell and immune checkpoint gene expression) showed higher proportions of CD3, CD8, CD20, FOXP3, CD68, and PDL1 IHC positively stained cells. Similarly, the high-ISG subtype (with increased interferon-stimulated gene expression and TP53 somatic mutations) had higher proportions of CD3, CD8, CD20, FOXP3, and CD68 stained cells than the low-TIL subtype and in particular exhibited high CD163 staining, indicative of a proinflammatory tumor microenvironment. CD8, CD20, CD163, PDL1, and IDO1 expression varied significantly by PAM50 subtypes, with luminal (A and B) subtypes showing lower staining than non-luminal (HER2 and Basal) subtypes. A distinctive feature, however, of luminal B tumors was the higher infiltration by CD163-expressing cells when compared with the luminal A subtype. Except for FOXP3, all examined IHC markers showed significantly higher expression in patients with somatic TP53 mutations (CD3: P= 0.003, CD8: P= 0.005, CD20: P= 0.003, CD68: P= 0.002, CD163: P= 0.002, PDL1: P= 0.003, and IDO1: P= 0.013). On the other hand, expression of these markers did not significantly vary by PIK3CA mutation or germline APOBEC3A/B deletion polymorphism, which is more prevalent among East Asians than other populations and was previously linked to BC risk and immune environment. Of the BC risk factors assessed, only body mass index (BMI) demonstrated associations with immune cell densities, with higher BMI associated with lower expression of CD3, CD8, and CD20. This study sheds further light on the in situ immune landscape of gene expression-based BC subtypes, suggesting that standard IHC assessment of immune markers could refine BC subtype classification for clinical and epidemiological use. Citation Format: Mustapha Abubakar, Hela Koka, Priscilla Lee, Scott Lawrence, Karun Mutreja, Difei Wang, Bin Zhu, Shelly Tse, Xiaohong R Yang. Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B016.