Dual Staining Research Articles

The molecular anti-metastatic potential of CBD and THC from Lebanese Cannabis via apoptosis induction and alterations in autophagy

The medicinal plant Cannabis sativa L. (C. sativa) is currently being extensively studied to determine the full extent of its therapeutic pharmacological potential. Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD) are the most thoroughly investigated compounds. We aimed to explore the anticancer activity of cannabinoids mixture isolated from the Lebanese C. sativa plant in ratios comparable to the local medicinal plant, to elucidate its mechanism of action in breast cancer cells in vitro. Cells were subjected to cytotoxicity assay, cell cycle analysis, Annexin V/PI dual staining, cell death ELISA, immunofluorescence, in addition to western blot analysis of apoptotic and autophagy markers. We further evaluated the anti-metastatic effect of cannabinoids on MDA-MB-231 using the scratch wound-healing, trans-well migration and invasion assays. Our results revealed the promising therapeutic benefits of CBD/THC on inhibiting the growth of breast cancer cells by promoting cellular fragmentation, phosphatidylserine translocation to the outer membrane leaflet and DNA fragmentation in both cell lines while inhibiting the motility of the triple negative breast cancer cells. In our study, CBD/THC mixture was found to exhibit a pro-apoptotic activity via the activation of the mitochondrial apoptotic pathway, independent from ROS production while also suggesting the activation of a caspase-dependent apoptotic pathway. Even though autophagy was altered upon exposure to the cannabinoid mixture, our data suggested that it is not the mechanism responsible of inducing cell death. In conclusion, our study demonstrates the promising therapeutic benefits of CBD and THC isolated from the Lebanese C. sativa plant on breast cancer cells in vitro.

Seasonal changes in the viability and abundance of bacterial cells in the snowpack ecosystem of a High Arctic ice cap

ABSTRACT Microbes play an essential role in nutrient turnover within Arctic environments, and their contribution to biogeochemical cycles can depend on several factors, including but not limited to cell viability. In this study, we employed the SYBR-PI dual cell stain to epifluorescence microscopy to enumerate proportions of potentially viable and non-viable bacterial cell populations within a melting snowpack on an ice cap, Foxfonna in Svalbard. Non-viable cells dominated on Foxfonna (2.5 ± 0.36 × 107 cells m−2) during the June to early July period, when biological production was usually at its peak. Furthermore, non-viable cells also dominated the total cell abundance within superimposed ice (223 ± 242 cells mL−1) and glacial ice (695 ± 717 cells mL−1) beneath the snow. We propose that the rapid, early loss of cell viability was caused by a number of abiotic and biotic factors. Hence, necromass (dead cell residue) contributed to the export of organic matter to downstream ecosystems.

Microbial and Transcriptomic Landscape Associated With Neutrophil Extracellular Traps in Perianal Fistulizing Crohn's Disease.

Perianal fistulizing Crohn's disease (pfCD) poses significant healing challenges, closely associated with neutrophil extracellular traps (NETs). This study aimed to investigate the microbe-host interactions influencing NETs in pfCD. From January 2019 to July 2022, patients with pfCD were screened at Ren Ji Hospital. Patients in remission following comprehensive treatment were recruited. We documented clinical characteristics, medication regimens, healing outcomes, and infliximab levels in fistula tissues. NET positivity was confirmed by positive results in citrullinated histone H3 (CitH3) enzyme-linked immunosorbent assay (ELISA) and dual immunofluorescence staining for myeloperoxidase and CitH3. Microbial and transcriptomic profiles from fistula tissues, obtained during surgery, were analyzed using 16S rRNA gene sequencing and RNA sequencing. Differences in microbiome and transcriptomic profiles were evaluated, and their relationships were assessed using Mantel's and Spearman's coefficients. Significant differences in microbial communities were found between groups (P = .007). Representatively differential microbes such as Prevotella bivia, Streptococcus gordonii, and Bacteroides dorei were enriched in NETs-positive fistulas (P < .05). Functional analysis of microbes revealed reduced ubiquinol biosynthesis and butanoate production in NETs-negative fistulas (P < .05). Transcriptomic analysis indicated increased neutrophil and monocyte infiltration in NETs-positive fistulas, associated with pathways involving bacterial response, neutrophil chemotaxis, secretory processes, and peptidase activity (P < .05). Species prevalent in NETs-positive fistulas correlated positively with immune responses and wound healing pathways, whereas bacteria in NETs-negative fistulas correlated negatively. NETs were negatively associated with tissue infliximab levels (P = .001) and healing outcomes (P = .025). Our findings reveal unique microbial and transcriptomic signatures associated with NETs in pfCD, highlighting their profound influence on clinical outcomes.

Protective Effects of Nettle Tea on SKOV-3 Ovarian Cancer Cells Through ROS Production, Apoptosis Induction, and Motility Inhibition Without Altering Autophagy.

Urtica dioica L. (UD), also known as the stinging nettle, has long been used in traditional medicine for its wide range of health benefits. The current study focuses on the effect of nettle tea on the growth and proliferation of one of the most aggressive ovarian adenocarcinoma cell line, SKOV-3 cells. To examine this, cytotoxicity, cell cycle analysis, and ROS assays were performed, along with Annexin V/PI dual staining, cell death ELISA, Western blot analysis, and motility assays. The results showed that a UD aqueous extract (UDAE) can inhibit the growth and proliferation of SKOV-3 cells in a dose- and time-dependent manner by promoting cellular fragmentation. This was accompanied by an increase in two apoptotic hallmarks, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation as revealed by cell death ELISA. This aqueous extract showed a pro-oxidant activity while also activating the extrinsic caspase-dependent apoptotic pathway with no alteration in autophagy markers. Furthermore, the extract showed promising inhibitory effect on the migratory capacities of aggressive ovarian cancer cells, in vitro.

Development of β‐Carboline‐Coumarin Based Hybrids as Potential Cytotoxic and Topoisomerase IIα Inhibitors

AbstractCancer remains a global health concern, prompting extensive efforts to develop novel inhibitors targeting the enzyme topoisomerase IIα as potential anti‐cancer agents. Herein, we strategically combined the pharmacophores from natural (β‐carboline) and (coumarin) sources, diversifying them at two distinct points. The in vitro evaluation of cytotoxic properties were estimated on human cancer cell lines, reveals that among the tested compounds, 13 r (isopropyl‐substituted β‐carboline) exhibited remarkable potency with IC50 value of 4.37 μM in the HCT116 cell line. Its selectivity for cancer cells was validated against normal keratinocyte cells, establishing a favourable selectivity index. Further, SAR studies indicate that no substitution (13 r) at C‐6 and C‐7 of β‐carboline, enhance anticancer activity. Assays confirmed its ability to intercalate with DNA, inhibit topoisomerase IIα, induce apoptosis, and disrupt mitochondrial membrane potential in HCT116 cells. Flow cytometric assays gauged the induction of apoptosis using Annexin‐V/PI dual staining and the disruption of mitochondrial membrane potential through JC staining. Results demonstrated that compound 13 r induced apoptosis and dose‐dependent depolarization of the mitochondrial membrane in HCT116 cells. Molecular docking studies further validated that compound 13 r binds within the active site of DNA when complexed with topo IIα, and this binding was stabilized through interactions with DNA base pairs and amino acid residues.

Thiazolidinedione-Conjugated Lupeol Derivatives as Potent Anticancer Agents Through a Mitochondria-Mediated Apoptotic Pathway.

To improve the potential of lupeol against cancer cells, a privileged structure, thiazolidinedione, was introduced into its C-3 hydroxy group with ester, piperazine-carbamate, or ethylenediamine as a linker, and three series of thiazolidinedione-conjugated compounds (6a-i, 9a-i, and 12a-i) were prepared. The target compounds were evaluated for their cytotoxic activities against human lung cancer A549, human breast cancer MCF-7, human hepatocarcinoma HepG2, and human hepatic LO2 cell lines, and the results revealed that most of the compounds displayed improved potency over lupeol. Compound 12i exhibited significant activity against the HepG2 cell line, with an IC50 value of 4.40 μM, which is 9.9-fold more potent than lupeol (IC50 = 43.62 μM). Mechanistic studies suggested that 12i could induce HepG2 cell apoptosis, as evidenced by AO/EB staining and annexin V-FITC/propidium iodide dual staining assays. Western blot analysis suggested that compound 12i can upregulate Bax expression, downregulate Bcl-2 expression, and activate the mitochondria-mediated apoptotic pathway. Collectively, compound 12i is worthy of further investigation to support the discovery of effective agents against cancer.

P53 ABNORMAL ENDOMETRIAL CANCERS – ANALYSIS OF PD-1 CO-EXPRESSION BY CD4 HELPER AND CD8 CYTOTOXIC T-CELL SUBTYPES IN THE TUMOR MICROENVIRONMENT

Abstract Introduction/Objective Endometrial cancer is classified into four molecular subtypes of which the p53 abnormal variant is biologically aggressive. Programmed cell death protein 1 (PD-1) is a cell surface receptor and a vital inhibitor of the immune response. Programmed death ligand 1 (PD-L1) is a trans-membrane co-inhibitory factor of the immune response. PD-1 / PDL-1 interaction inhibits the activation and survival of T cells and decreases cytokine secretion. Some cancer cells express PD-L1, which then bind to PD-1 expressed on tumor specific T cells, and escape destruction by the T cells. The CD4/CD8 T cell immune response in the p53 abnormal variant has not been adequately evaluated with respect to the PD-1/PD-L1 pathway. Understanding this immune microenvironment is important to assess the potential use of anti-PD-1 targeted tumor therapy. Methods/Case Report Twenty cases of p53 abnormal endometrial cancers were obtained. All cases were PDL-1 +ve by immunohistochemistry. Two dual stains were performed - PD-1/CD4 and PD-1/CD8 antibodies. Six high-power fields (40x objective) with the greatest concentration of lymphocytes were counted. The total number of CD4 or CD8-only cells was recorded for each of the two stained slides for each case. The total number of PD- 1/CD4 and PD-1/CD8 positive cells were counted and recorded. The results were tabulated, and a paired t-test statistical analysis was performed to compare the total number of CD4 and CD8 cells and the percentage of PD-1/CD4 cells with the percentage of PD-1/CD8 positive cells. Results (if a Case Study enter NA) A statistically significant increase in the number of CD8 (Mean = 383) compared to CD4 cells (Mean = 317) was seen in the tumor microenvironment (p-value=0.0023). Evaluating PD-1 expression, the converse was true, and the percentage of PD-1/CD4 cells (Mean = 13.035%) was increased compared to the percentage of PD-1/CD8 cells (Mean = 9.205%), achieving statistical significance (p-value=0.0116). Conclusion The statistically significant increase in CD8 cells in the tumor and its stroma suggests that a robust cytotoxic response is being mounted to this high-risk molecular variant. The concurrent increase in CD4/PD-1 positive lymphocytes provides an appropriate microenvironment for the use of anti-PD-1 immunotherapy, since blockade of PD-1 on CD4 helper T cells may augment the tumor ablative cytotoxic properties of the CD8 positive lymphocytes. This includes reinvigoration of exhausted CD8+ T cells. Anti-PD-1 may also prevent the inhibition of PD-1 positive CD8 cells.

Anticancer potential of isoalantolactone in testicular cancer: an analysis of cytotoxicity, apoptosis, and signaling pathways.

Testicular cancer, a highly prevalent malignancy among young adults, has witnessed an alarming rise in recent decades. This study delves into the therapeutic potential of isoalantolactone (IATL), a natural product extracted from Inula helenium and Inula racemosa, against testicular cancer. Employing MTT assays and flow cytometry, we observed a dose-dependent reduction in cell viability and induction of cell cycle arrest at sub-G1 phase with increasing IATL concentrations. Furthermore, Annexin V/PI dual staining revealed IATL-induced apoptosis. Human Apoptosis Array analysis demonstrated IATL's influence on HIF-1α and TNF R1 expression, implicating its role in cancer cell growth and death regulation. Next-generation sequencing (NGS) and pathway analysis highlighted the involvement of ferroptosis and HIF-1 signaling in IATL-mediated effects. Western blotting validated the downregulation of key proteins associated with apoptosis inhibition and activation, confirming IATL's potential as an anticancer agent. Moreover, IATL induced ferroptosis by modulating expression levels of GPX4, xCT, NRF2, and HO-1. Our findings shed light on IATL's multifaceted anticancer mechanisms, emphasizing its potential as a therapeutic candidate for testicular cancer.

Breast and axillary marking in the neoadjuvant setting: survey results from experts of the Brazilian society of mastology.

The precise location of the tumor site is essential for the success of surgical treatment. Neoadjuvant chemotherapy (NAC) is a challenge for preoperative tumor and node localization. Thus, the knowledge and attitudes of the affiliated members of the Brazilian Society of Mastology (SBM) regarding breast and axilla marking were evaluated and a consensus regarding management and treatment was reached. This was an online survey conducted between June and December 2022. All 1,742 active mastologists affiliated to the SBM were invited anonymously. The online form contained 28 objective questions, of which 22 were formulated on a Likert scale. These questions addressed relevant aspects related to breast and axilla marking in the neoadjuvant setting. Responses that reached 70% agreement were considered consensual. Statistical analysis was performed using the SPSS program version 26.0. Post hoc analysis was performed when appropriate and the significance level was set at p < 0.05. Polychoric regression analyses were conducted using `VGAM` package. In total, 468 mastologists answered the questionnaire (26.8%), with a predominance of professionals aged between 40-49 years (32.1%). Most professionals were board-certified (84,8%). The indication of tumor marking in the breast prior to NAC was consensual (96.4%) and the metal clip was the preferred method (69.7%). There was no consensus regarding the indication of pre-NAC histologically positive lymph node marking (49.8% disagree and 42.8% agree). However, there was consensus that the clinical and imaging evaluation was insufficient for staging the axilla as N1 (71.6%). The contraindication of breast and node marking in T4b tumors (71.2%) was consensual. There was consensus on the indication of sentinel lymph node biopsy (SLNB) for initially cN1 (92.3%) or cN2 (72.7%) tumors that became cN0 after NAC, with 67.5% opting for dual staining with technetium and patent blue. When <3 lymph nodes were retrieved 41.0% of mastologists performed axillary lymphadenectomy. Among the 28 questions, consensus was reached on only 11 (39.3%). The indication of pre-NAC breast marking is consensual among Brazilian mastologists, although axillary nodal marking is not. There is a great divergence of attitudes among Brazilian surgeons in relation to the many issues related to pre-NAC breast and axilla marking.

Distribution of 14 High-Risk HPV Types and p16/Ki67 Dual-Stain Status in Post-Colposcopy Histology Results: Negative, Low- and High-Grade Cervical Squamous Intraepithelial Lesions.

Determining the distribution of high-risk human papillomavirus (HR-HPV) types in histologic low-(LSIL) and high-grade (HSIL/CIN2+) squamous intraepithelial lesions through a diagnostic process in a cervical cancer prevention provides one of the key etiological factors behind further progression and persistence. Incorporating novel high-grade cervical lesion biomarkers such as p16/Ki67 dual staining (DS) alongside HPV typing has become important in detecting cervical precancers. Among 28,525 screening tests and 602 histology results, 559 cases with HR-HPV and histology results obtained from colposcopic biopsy were retrospectively analyzed, together with DS status. The χ2 test with Bonferroni correction evaluated the differences in HR-HPV type prevalence and DS positivity across three histologic study groups. A statistically significant difference in the prevalence of HPV 16 was observed between negative and HSIL/CIN2+ (p = 0.00027) groups, as well as between the LSIL/CIN1 and HSIL/CIN2+ groups (p = 0.00041). However, no significant difference was found between the negative and LSIL/CIN1 groups. Similarly, the DS positivity difference was significant between the negative and HSIL/CIN2+ (p < 0.0001) and between the LSIL/CIN1 and HSIL/CIN2+ groups (p < 0.0001), but there was no significant difference between the negative and LSIL/CIN1 groups. The study highlights the heterogeneous nature of HPV-related cervical pathologies, and the distinct risks associated with different cervical lesion grades, emphasizing the importance of HR-HPV type distribution and DS status.

Antiproliferative Effects of Methanolic Fruit Extract of Solanum xenthocarpum (L.) on Human Breast Cancer Cells.

Solanum xanthocarpum, a perennial herb native to India, contains steroidal glycoalkaloids with notable anticancer properties. This study investigated the antioxidant and antiproliferative effects ofmethanolic fruit extract of S. xanthocarpum on human breast cancer cells (MDA-MB-231). Phytochemical screening and LC-HRMS analysis confirmedpresence of various primary and secondary metabolites. Antioxidant activity was assessed through DPPH, ABTS radical scavenging, reducing power, and phosphomolybdate assays. The extract demonstrated significant antioxidant potential with EC50 values of 60.10 ± 0.88 µg/mL (DPPH) and 392.29 ± 3.93 µg/mL (ABTS). Cytotoxicity against MDA-MB-231 cells was evaluated via morphological analysis, MTT assays, and IC50 determination (24.19 ± 0.56 µg/L). Apoptosis was confirmed using dual staining techniques (AO/EB, Hoechst 33342/PI, DAPI), revealing condensed nuclei, apoptotic bodies, and reduced mitochondrial membrane potential, as indicated by Rhodamine staining. Additionally, increased reactive oxygen species (ROS) levels were observed using H2-DCF-DA staining. The total phenolic and flavonoid contents of the extract were 127.78 ± 3.547 mg GAE/g and 98.06 ± 4.289 mg QE/g, respectively. These findings suggest that the methanolic fruit extract of S. xanthocarpum possesses strong antioxidant and anticancer activities, indicating its potential role in cancer treatment. Further studies are warranted to explore its bioactive compounds for developing novel anticancer therapies.

Cost-effectiveness analysis of alternative screening strategies for the detection of cervical cancer among women in rural areas of Western Kenya.

While the incidence of cervical cancer has dropped in high-income countries due to organized cytology-based screening programs, it remains the leading cause of cancer death among women in Eastern Africa. Therefore, the World Health Organization (WHO) now urges providers to transition from widely prevalent but low-performance visual inspection with acetic acid (VIA) screening to primary human papillomavirus (HPV) DNA testing. Due to high HPV prevalence, effective triage tests are needed to identify those lesions likely to progress and so avoid over-treatment. To identify the optimal cost-effective strategy, we compared the VIA screen-and-treat approach to primary HPV DNA testing with p16/Ki67 dual-stain cytology or VIA as triage. We used a Markov model to calculate the budget impact of each strategy with incremental quality-adjusted life years and incremental cost-effectiveness ratios (ICER) as the main outcome. Deterministic cost-effectiveness analyses show that the screen-and-treat approach is highly cost-effective (ICER 2469 Int$), while screen, triage, and treat with dual staining is the most effective with favorable ICER than triage with VIA (ICER 9943 Int$ compared with 13,177 Int$). One-way sensitivity analyses show that the results are most sensitive to discounting, VIA performance, and test prices. In the probabilistic sensitivity analyses, the triage option using dual stain is the optimal choice above a willingness to pay threshold of 7115 Int$ being cost-effective as per WHO standards. The result of our analysis favors the use of dual staining over VIA as triage in HPV-positive women and portends future opportunities and necessary research to improve the coverage and acceptability of cervical cancer screening programs.

Alpinia galanga induces caspase-dependent apoptotic cell death in human lung and cervical cancer cells

Introduction: Alpinia galanga (L.) Willd is a rhizomatous, recurrent herb used to treat numerous diseases, including cancer. This study examines the efficiency of the different parts of A. galanga for their phytochemical, antioxidant, and anti-cancer properties. Methods: The powdered leaf, stem, and rhizome of A. galanga were extracted using hexane, ethyl acetate and methanol; the phytochemical compositions of the extracts were characterized using high performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS). Their antioxidant potentials were evaluated using different assays, including 2,2-Diphenyl-1picrylhydrazil (DPPH), 2-Azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), Superoxide anion (O2-), Hydroxyl radical (OH), and Phosphomolybdenum assays. The in vitro anticancer activity was studied using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Comet assay was used to determine the level of DNA damage. Inductions of morphological alterations were studied using the AO/Et-Br dual staining method. The ELISA kits were used to measure caspase activities. Results: The cytotoxicity results showed that the various extracts had inhibitory and cytotoxic effects on cancer cell lines. The leaf hexane (LH) extract of A. galanga induced DNA damage with prominent increased tail length and tail moment followed by the induction of apoptotic cells and up-regulation of the caspase activities. HPLC and GC-MS analysis allowed the identification of bioactive compounds, recognized as apoptotic inducers in human cancer cells by activating caspase-dependent pathways. Conclusion: This work reports for the first time the effect of LH extract of A. galanga in triggering apoptosis in A-549 and HeLa cells through the upregulation of caspase-8 and caspase-3 activities.

Concanavalin: A Loaded Chitosan Nanoparticles Synthesis and Characterization to Inhibit the Proliferation of Lung Cancer

Background Non-small-cell lung cancer is the most ubiquitous malignancy, and the present treatments do not offer the most effective outcomes for therapy. Natural drug therapy, which directly targets tumor cells, is now recognized as a safe technique for cancer treatment. The use of tailored nanoparticles also improves the efficacy of cancer therapy. Purpose To synthesize, characterize, and evaluate the natural drug concanavalin A (ConA) coated with chitosan nanoparticles (ConA-CS NPs) for cytotoxicity against lung cancer cell line (NCI-H358). Methods The current study demonstrates the development of ConA-CS NPs as well as their unique characteristics, anticancer processes, and possible applications in treatment. Using UV–Vis, Fourier transform infrared, X-ray diffraction, field emission scanning electron microscopy, and dynamic light scattering studies, ConA-CS NPs were generated and evaluated. The mitochondrial toxicity test assay was used to examine ConA-CS NP-induced cytotoxicity. Dual (acridine orange/ethidium bromide) staining was performed to examine apoptotic modifications, and mitochondrial membrane potential levels in NCI-H358 cells were examined using the appropriate fluorescence staining assays. Results and Conclusion The use of ConA-CS NPs for enhanced tumor treatment, which combines natural drugs to eradicate the tumor and prevent relapses, is supported by in vitro investigations. The ability of ConA-encapsulated CS NPs to impede human lung carcinoma cell growth has not yet been investigated. Therefore, this investigation may provide a new paradigm for cancer detection and therapy, and the results may surely help to enhance the quality of life for cancer patients.

Assessing anticancer properties of PEGylated platinum nanoparticles on human breast cancer cell lines using in-vitro assays

This study describes the in-vitro cytotoxic effects of PEG-400 (Polyethylene glycol-400)-capped platinum nanoparticles (PEGylated Pt NPs) on both normal and cancer cell lines. Structural characterization was carried out using x-ray diffraction and Raman spectroscopy with an average crystallite size 5.7 nm, and morphological assessment using Scanning electron microscopy (SEM) revealed the presence of spherical platinum nanoparticles. The results of energy-dispersive x-ray spectroscopy (EDX) showed a higher percentage fraction of platinum content by weight, along with carbon and oxygen, which are expected from the capping agent, confirming the purity of the platinum sample. The dynamic light scattering experiment revealed an average hydrodynamic diameter of 353.6 nm for the PEGylated Pt NPs. The cytotoxicity profile of PEGylated Pt NPs was assessed on a normal cell line (L929) and a breast cancer cell line (MCF-7) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results revealed an IC50 of 79.18 μg ml−1 on the cancer cell line and non-toxic behaviour on the normal cell line. In the dual staining apoptosis assay, it was observed that the mortality of cells cultured in conjunction with platinum nanoparticles intensified and the proliferative activity of MCF-7 cells gradually diminished over time in correlation with the increasing concentration of the PEGylated Pt NPs sample. The in vitro DCFH-DA assay for oxidative stress assessment in nanoparticle-treated cells unveiled the mechanistic background of the anticancer activity of PEGylated platinum nanoparticles as ROS-assisted mitochondrial dysfunction.

Shilajit (Mumio) Elicits Apoptosis and Suppresses Cell Migration in Oral Cancer Cells Through Targeting Urokinase-type Plasminogen Activator and Its Receptor and Chemokine Signaling Pathways

Background Shilajit a natural phytomineral has a proven record of treating many human body ailments. Purpose This study aimed to explore the influence of Shilajit on cell proliferation and apoptosis induction in oral cancer cells (OCC) and to comprehend the molecular mechanism associated with OCC migration. Materials and Methods Human gingival fibroblast (hGF) and OCC (KB-1 (KERATIN-forming tumor cell line HeLa)) were exposed to Shilajit solution dilutions. Cell growth and apoptosis were measured by MTT and Annexin-V tests (control/test group). Cellular morphology using an inverted microscope, cellular apoptosis using acridine orange/ethidium bromide dual staining, reactive oxygen species production analysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and gene protein expression using real-time polymerase chain reaction test were used to measure study outcomes. The data enumerated were the average of three trial experiments. Categorical and numerical data were expressed as frequency distribution and means, respectively. Differences in groups (control/test; zero/24 h/48 h) were determined using Student’s t-test and one way analysis of variance, with probability ‘ p’ value considered significant at ≤0.05. Results The viability of OCC exhibited a concentration and time-dependent response to Shilajit. Notably, Shilajit demonstrated selectivity against cancer cells. Through an examination of the Annexin-V apoptosis assay, it was observed that Shilajit induces apoptosis by upregulating the proapoptotic gene expression ( p ≤ 0.05) and downregulating antiapoptotic proteins ( p ≤ 0.05). Furthermore, the impact of Shilajit on cell migration decreased significantly when compared to control cells through modulating the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) and chemokines gene expression. Conclusion Shilajit exhibited greater cytotoxicity, decreased OCC proliferation and migration, and initiated OCC apoptosis as equivalent to normal cells. These promising outcomes indicate that Shilajit holds potential as a robust, promising option for oral cancer treatment, underscoring the need for further research in this domain.

Functional Identification and Regulatory Active Site Screening of the DfDXS Gene of Dryopteris fragrans.

Dryopteris fragrans (L.) Schott has anti-inflammatory and antioxidant properties, and terpenoids are important components of its active constituents. The methyl-D-erythritol 4-phosphate (MEP) pathway is one of the major pathways for the synthesis of terpene precursors in plants, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in this pathway. DXS has been shown to be associated with increased stress tolerance in plants. In this experiment, two DXS genes were extracted from the D. fragrans transcriptome and named DfDXS1 and DfDXS2. Based on phylogenetic tree and conserved motif analyses, DXS was shown to be highly conserved evolutionarily and its localization to chloroplasts was determined by subcellular localization. Prokaryotic expression results showed that the number and growth status of recombinant colonies were better than the control under 400 mM NaCl salt stress and 800 mM mannitol-simulated drought stress. In addition, the DfDXS1 and DfDXS2 transgenic tobacco plants showed improved resistance to drought and salt stress. DfDXS1 and DfDXS2 responded strongly to methyl jasmonate (MeJA) and PEG-mimicked drought stress following exogenous hormone and abiotic stress treatments of D. fragrans. The transcriptional active sites were investigated by dual luciferase and GUS staining assays, and the results showed that the STRE element (AGGGG), the ABRE element (ACGTGGC), and the MYC element (CATTTG) were the important transcriptional active sites in the promoters of the two DXS genes, which were closely associated with hormone response and abiotic stress. These results suggest that the DfDXS gene of D. fragrans plays an important role in hormone signaling and response to stress. This study provides a reference for analyzing the molecular mechanisms of stress tolerance in D. fragrans.

Metal complexes containing vitamin B6-based scaffold as potential DNA/BSA-binding agents inducing apoptosis in hepatocarcinoma (HepG2) cells.

A ligand (HL) was synthesized from the pyridoxal hydrochloride (vitamin B6 form) and 1-(2-Aminoethyl)piperidine in one single step. The metal complexes [Zn(L)(Bpy)]NO3 (1), [Cu(L)(Bpy)]NO3 (2), and [Co(L)(Bpy)]NO3 (3) were prepared by tethering HL and 2,2'-bipyridine. The synthesized HL and metal complexes 1-3 were thoroughly characterized using spectroscopic techniques such as 1H NMR, 13C NMR, FTIR, EI-MS, molar conductance, and magnetic moment, in addition to CHN elemental analysis. The geometry of complexes was square pyramidal around the metal ions {Zn(II), Cu(II), and Co(II)}. The interaction of ligand and metal complexes with DNA and BSA macromolecules was accomplished by UV-Vis absorption and fluorescence spectroscopy in vitro. The hyperchromism in band at 303-325 with no shift supports the groove binding with some partial intercalation in grooves. Similarly, in BSA-binding studies, complex 2 shows greater binding potential in the hydrophobic core probably near the Trp-212 in the subdomain IIA. Furthermore, complex 2 shows excellent cytotoxicity on HepG2 cancer cells with IC50 = 25.0 ± 0.45µM. The detailed analysis by cell-cycle studies shows cell arrest at the G2/M phase. The type of cell death was authenticated by an annexin V-FTIC dual staining experiment that reveals maximum death by apoptosis together with non-specific necrosis.

Development of Amphotericin B Decorated Gold Nanoparticles as a Promising Antileishmanial Nanoconjugate.

Leishmaniasis, attributed to the protozoan parasite Leishmania, manifests in diverse clinical forms, including cutaneous, mucocutaneous, and visceral leishmaniasis; VL constitutes a significant global health menace. Prevalent in tropical and subtropical regions, this affliction disproportionately impacts individuals below the poverty threshold, transmitted through the bite of female sandflies. Existing treatments, such as pentavalent antimony, miltefosine, and Amphotericin B, exhibit limitations. Despite the emergence of liposomal Amphotericin B (AmBisome) as a promising antileishmanial agent, its utility is impeded by adverse effects, elevated production expenses, and cytotoxicity. To address these challenges, our investigation introduces a potential remedy─a citrate-coated gold Amphotericin B nanoparticle formulation. Characterized using dynamic light scattering and transmission electron microscopy, this pioneering formulation exhibited efficacy against L. donovani Ag83 promastigotes as demonstrated by MTT cell viability testing. Evaluating internal reactive oxygen species (ROS) levels and dual staining with acridine orange and ethidium bromide unveiled its consequential impact on cell death. Significantly, our study discloses this novel nanoformulation's unprecedented inhibition of the trypanothione reductase enzyme. The findings posit the citrate-coated gold Amphotericin B nanoformulation as a promising and targeted antileishmanial agent, representing potential advancements in leishmaniasis therapeutics.

Mucoadhesive and drug release of cholic acid based thiomeric micelles and encapsulated silver and gold nanoparticles for anticancer studies

Among the various drug delivery system, mucoadhesive formulation is a promising approach to deliver drugs and vaccine through close contact with the absorption site and mucous membrane. Bile acids have received considerable interest in drug delivery (DD) applications owing to their advantages of biocompatibility, ability to act as both drug solubilizing and permeation-modifying agents. In this aspects, cholic acid (CA) based thiomeric micelles with improved mucoadhesive properties have been synthesized by functionalization of thiol groups and conjugation of PEG. The synthesized CA based thiomeric micelles (TCA-PEG) were characterized by 1HNMR, ATR-IR, GPC, Zeta potential and contact angle techniques. The critical micelle concentration (CMC) of TCA-PEG was measured by a fluorescence spectrometer using pyrene as a probe. The mucoadhesive properties of TCA-PEG polymeric micelles (PMs) were examined on the goat anterior tongue mucosa with an effectively higher adhesion of 40 % up to 3 h study period. Also, curcumin loaded TCA-PEG PMs showed only 13 % of the drug released up to the period of 3 h. The TCA-PEG PMs act as reducing and stabilizing agents for the preparation of gold and silver nanoparticle (Ag and Au NPs) and were characterized by UV–visible, DLS, Zeta potential, XPS, and HR-TEM techniques. The cytotoxicity and cellular apoptosis of TCA-PEG PMs encapsulated Ag and Au NPs were examined by MTT assay and AO/PI dual staining using MCF-7 breast cancer cells wherein AgNPs showed an effective cytotoxicity than AuNPs. The developed TCA-PEG PMs act as reservoir for soubilization of hydrophobic and hydrophilic drugs and ideal template for encapsulation of silver and gold nanoparticles for mucoadhesive DD system.