The Role of Vimentin in Human Corneal Fibroblast Spreading and Myofibroblast Transformation.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFß-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFß had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, ß1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

Divergent synthesis of complex withanolides enabled by a scalable route and late-stage functionalization.

Withanolides are a group of naturally occurring C28 steroids based on an ergostane skeleton. They have a high degree of polyoxygenation, and the abundance of O-functional groups has enabled various natural alterations to both the carbocyclic skeleton and the side chain. Consequently, these molecules have intricate structural features that lead to their highly varied display of biological activities including anticancer, anti-inflammatory, and immunomodulating properties. Despite being intriguing leads for further discovery research, synthetic access to the withanolides remains highly challenging-compounds for current biological research are mainly isolated from plants, often inefficiently. Here, we report the divergent synthesis of 11 withanolides in 12 to 20 steps, enabled by a gram-scale route and a series of late-stage functionalizations, most notably a bioinspired photooxygenation-allylic hydroperoxide rearrangement sequence. This approach enables further biological research disconnected from a reliance on minute quantities of the parent natural products or their simple derivatives.

Withaferin A alleviates inflammation in animal models of arthritis by inhibiting the NF-κB pathway and cytokine release.

Withaferin A, a steroid lactone from Withania somnifera, exhibits anti-inflammatory, immunomodulatory, and antioxidant properties. This study investigated the effects of withaferin A on collagen-induced arthritis (CIA) rats, focusing on NF-κB p65 regulation and cytokine release. Withaferin A (50 mg/kg b.wt., orally) or methotrexate (0.25 mg/kg b.wt., i.p., as a reference drug) was given to CIA rats daily for 20 days postarthritis induction. Joints were removed from nonarthritic and arthritic rats to assess the levels of NO, MPO, interleukin (IL)-1ß, IL-6, IL-10, TNF-α, COX-2, and NF-κB via ELISA. Furthermore, the mRNA expression of IL-1ß, IL-10, TNF-α, COX-2, iNOS, and NF-κB was also assessed through qPCR. Treatment with withaferin A significantly inhibited the levels of inflammatory cytokines and the transcription factor NF-κB; suppressed the expression of IL-1ß, IL-10, TNF-α, COX-2, iNOS, and NF-κB in the joint tissue of CIA rats; and reduced cartilage and bone destruction, as shown by H&E staining. To confirm the results obtained from biochemical and molecular studies and to determine the molecular target of withaferin A, we performed a molecular simulation of the potential targets of withaferin A, which identified the NF-κB pathway as its target. These results suggested that withaferin A effectively attenuated rheumatoid arthritis progression by inhibiting the activation of the NF-κB pathway and the downstream secretion of inflammatory cytokines.

In vivo evaluation of efficacy and safety of Coagulansin-A in treating arthritis.

The current study aimed to determine the safety and efficacy of Coag-A through in vivo analysis in CFA induced mice model. Treatment of CFA induced arthritis in mice with Coagulansin-A (10 mg/kg i.p. daily for 28 days), a withanolide obtained from Withania coagulans, as well as standard drug treatment with Dexamethasone (5 mg/kg i.p) was provided. The effect of Coag-A on body weight, relative organ weight, hematology, serum biochemistry, survival rate, oxidative stress markers, and antioxidant enzymes was evaluated. The liver and kidney histopathology were also assessed to ascertain its safety profile. Treatment of arthritic mice with Coag-A considerably improved body weight, relative organ weight of liver, kidney, and spleen, ameliorated hematology and serum biochemistry, and increased survival and antioxidant potential. Coag-A was found to be safer with fewer adverse effects showing hepato-protective, nephroprotective, and anti-inflammatory effect. It also significantly (p < 0.001) improved histopathology of CFA-induced mice when compared with Dexa. In conclusion, compared to dexamethasone, Coag-A has demonstrated a greater therapeutic benefit and fewer side effects in the treatment of arthritis against the CFA-induced model.

Development, characterization, and evaluation of withaferin-A and artesunate-loaded pH-responsive acetal-dextran polymeric nanoparticles for the management of malaria.

Artemisinin and its derivatives have been commonly used to treat malaria. However, the emergence of resistance against artemisinin derivatives has posed a critical challenge in malaria management. In the present study, we have proposed a combinatorial approach, utilizing pH-responsive acetal-dextran nanoparticles (Ac-Dex NPs) as carriers for the delivery of withaferin-A (WS-3) and artesunate (Art) to improve treatment efficacy of malaria. The optimized WS-3 and Art Ac-Dex NPs demonstrated enhanced pH-responsive release profiles under parasitophorous mimetic conditions (pH 5.5). Computational molecular modeling reveals that Ac-Dex's polymeric backbone strongly interacts with merozoite surface protein-1 (MSP-1), preventing erythrocyte invasion. In-vitro antimalarial activity of drug-loaded Ac-Dex NPs reveals a 1-1.5-fold reduction in IC50 values compared to pure drug against the 3D7 strain of Plasmodium falciparum. Treatment with WS-3 Ac-Dex NPs (100 mg/kg) and Art Ac-Dex NPs (30 mg/kg) to Plasmodium berghei-infected mice resulted in 78.11 % and 100 % inhibition of parasitemia. Notably, the combination therapy comprised of Art and WS-3 Ac-Dex NPs achieved complete inhibition of parasitemia even at a half dose of Art, indicating the synergistic potential of the combinations. However, further investigations are necessary to confirm the safety and effectiveness of WS-3 and Art Ac-Dex NPs for their successful clinical implications.

Withanolide Profile and Acetylcholinesterase Inhibitory Activity of Two Argentinean Jaborosa Species.

Acetylcholinesterase (AChE) inhibitors are still an important option for managing symptoms of mild to moderate Alzheimer's disease. In this study, we aimed to evaluate the potential in vitro AChE inhibitory activity of two Argentinian endemic Solanaceae species, Jaborosa bergii and J. runcinata. UHPLC-DAD-HRMS metabolite profiling revealed the presence of withanolides in the active CH2Cl2 subextracts. Their fractionation led to the isolation and identification of two known spiranoid withanolides from J. runcinata and three new withanolides with a skeleton similar to that of trechonolide-type withanolides from J. bergii. The known compounds showed moderate AChE inhibitory activity, while the new ones were inactive.

Withanolides production by the endophytic fungus Penicillium oxalicum associated with Withania somnifera (L.) Dunal.

Withanolides are steroidal lactones with diverse bioactive potential and their production from plant sources varies with genotype, age, culture conditions, and geographical region. Endophytic fungi serve as an alternative source to produce withanolides, like their host plant, Withania somnifera (L.) Dunal. The present study aimed to isolate endophytic fungi capable of producing withanolides, characterization and investigation of biological activities of these molecules. The methanolic fungal crude extract of one of the fungal isolates WSE16 showed maximum withanolide production (219 mg/L). The fungal isolate WSE16 was identified as Penicillium oxalicum based on its morphological and internal transcribed spacer (ITS) sequence analysis and submitted in NCBI (accession number OR888725). The methanolic crude extract of P. oxalicum was further purified by column chromatography, and collected fractions were assessed for the presence of withanolides. Fractions F3 and F4 showed a higher content of withanolides (51.8 and 59.1 mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC50 of 23.52 and 17.39 µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC50 of 39.42 and 38.71 µg/ml, superoxide anion scavenging assay with IC50 of 41.10 and 38.84 µg/ml, and reducing power assay with IC50 of 42.61 and 41.40 µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC50 of 30.34 and 22.05 µg/ml, respectively). The withanolides present in fraction 3 and fraction 4 were identified as (20S, 22R)-1a-Acetoxy-27-hydroxywitha-5, 24-dienolide-3b-(O-b-D-glucopyranoside) and withanamide A, respectively, using UV, FTIR, HRMS, and NMR analysis. These results suggest that P. oxalicum, an endophytic fungus isolated from W. somnifera, is a potential source for producing bioactive withanolides.

Withaferin A Inhibits Liver Cancer Tumorigenesis by Suppressing Aerobic Glycolysis through the p53/IDH1/HIF-1α Signaling Axis.

BACKGROUND: The energy supply of certain cancer cells depends on aerobic glycolysis rather than oxidative phosphorylation. Our previous studies have shown that withaferin A (WA), a lactone compound derived from Withania somnifera, suppresses skin carcinogenesis at least partially by stabilizing IDH1 and promoting oxidative phosphorylation. Here, we have extended our studies to evaluate the anti-tumor effect of WA in liver cancer. METHODS: Differential expression of glycolysis-related genes between liver cancer tissues and normal tissues and prognosis were verified using an online database. Glycolysis-related protein expression was detected using western blot after overexpression and knockdown of IDH1 and mitochondrial membrane potential assay based on JC-1, and mitochondrial complex I activity was also detected. The inhibitory effect of WA on the biological functions of HepG2 cells was detected along with cell viability using MTT assay, scratch assay, clone formation assay, glucose consumption and lactate production assay. Western blot and qRT-PCR were used to detect the expression of proteins and genes related to IDH1, p53 and HIF1α signaling pathways. RESULTS: We first identified that IDH1 expression was downregulated in human liver cancer cells compared to normal liver cells. Next, we found that treatment of HepG2 cells with WA resulted in significantly increased protein levels of IDH1, accompanied by decreased levels of several glycolytic enzymes. Furthermore, we found that WA stabilized IDH1 proteins by inhibiting the degradation by the proteasome. The tumor suppressor p53 was also upregulated by WA treatment, which played a critical role in the upregulation of IDH1 and downregulation of the glycolysis-related genes. Under hypoxic conditions, glycolysis-related genes were induced, which was suppressed by WA treatment, and IDH1 expression was still maintained at higher levels under hypoxia. CONCLUSION: Taken together, our results indicated that WA suppresses liver cancer tumorigenesis by p53-mediated IDH1 upregulation, which promotes mitochondrial respiration, thereby inhibiting the HIF-1α pathway and blocking aerobic glycolysis.

Leptin combined with withaferin A boost posthemorrhagic neurogenesis via activation of STAT3/SOCS3 pathway.

Neurogenesis as a potential strategy to improve the consequences of intracerebral hemorrhage (ICH). The current study investigates the effects of withaferin A (WFA) in combination with leptin (LEP) on ICH and neurogenesis mechanisms. LEP levels were dramatically reduced on days 7 and 14 following ICH insults in mice, but continuous WFA therapy significantly improved the potency of intrinsic LEP on day 14 after ICH. Furthermore, WFA combined with LEP enhances intrinsic neurogenesis and lessen motor deficits and long-term cognitive outcomes after ICH. In parallel, leptin deficiency in ob/ob mice limits enhancement of neurogenesis following ICH in response to WFA combined with LEP treatment. Importantly, the functional recovery conferred by WFA combined with LEP after ICH was inhibited by neurogenesis suppression. Mechanistically, this study unveiled that the signal transducer and activator of transcription-3 (STAT3) / suppressor of cytokine signaling-3 (SOCS3) pathway is a critical signaling pathway through which WFA combined with LEP treatment promotes intrinsic neurogenesis after ICH. Collectively, the results of this study elucidate the neuroprotective effects of WFA and LEP in ICH, and highlight a potential approach for ICH cell therapy.

Withania frutescens (L.) Pauquy, a valuable Mediterranean shrub containing bioactive withanolides.

The bushy plant Withania frutescens (L.) Pauquy is well distributed in the West-Mediterranean area, notably in the south of Spain, Algeria and Morocco where is it is used traditionally for the treatment of various human diseases, including diabetes. Unlike the two major species W. somnifera and W. coagulans extensively studied, the genomically close species W. frutescens has been much less investigated. Nevertheless, this shrub species displays a comparable phytochemical profile and marked antioxidant and anti-inflammatory properties, at the origin of reported pharmacological effects and its traditional uses. Here we have analyzed the diversity of biological effects reported with leaves and root extracts of W. frutescens. Hydroalcoholic extracts prepared from the aerial parts of the plant have revealed antihyperglycemic and cell-protective activities along with antimicrobial and anticorrosive effects. The extracts contained diverse polyphenolic compounds and a few alkaloids (calystegines) but most of the observed effects have been attributed to the presence of withanolides which are modified C28 ergostane-type steroids. Our analysis focused in part on specific withanolides found in W. frutescens, in particular an unusual 3-O-sulfated withanolide considered as a potential pro-drug of the major active compound withaferin A (WA) and a lead compound for the development of a potential drug candidate. The mechanism of action of this sulfated WA analogue is discussed. Altogether, our unprecedented extensive analysis of W. frutescens highlighted the pharmacological potential of this atypical medicinal plant. By analogy with the major cultivated Withania species, the market potential of little-known plant is underlined.

A novel combinatorial approach using sulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms.

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia.

In our previous studies, we showed that the generation of ovarian tumors in NSG mice (immune-compromised) resulted in the induction of muscle and cardiac cachexia, and treatment with withaferin A (WFA; a steroidal lactone) attenuated both muscle and cardiac cachexia. However, our studies could not address if these restorations by WFA were mediated by its anti-tumorigenic properties that might, in turn, reduce the tumor burden or WFA's direct, inherent anti-cachectic properties. To address this important issue, in our present study, we used a cachectic model induced by the continuous infusion of Ang II by implanting osmotic pumps in immunocompetent C57BL/6 mice. The continuous infusion of Ang II resulted in the loss of the normal functions of the left ventricle (LV) (both systolic and diastolic), including a significant reduction in fractional shortening, an increase in heart weight and LV wall thickness, and the development of cardiac hypertrophy. The infusion of Ang II also resulted in the development of cardiac fibrosis, and significant increases in the expression levels of genes (ANP, BNP, and MHCß) associated with cardiac hypertrophy and the chemical staining of the collagen abundance as an indication of fibrosis. In addition, Ang II caused a significant increase in expression levels of inflammatory cytokines (IL-6, IL-17, MIP-2, and IFNγ), NLRP3 inflammasomes, AT1 receptor, and a decrease in AT2 receptor. Treatment with WFA rescued the LV functions and heart hypertrophy and fibrosis. Our results demonstrated, for the first time, that, while WFA has anti-tumorigenic properties, it also ameliorates the cardiac dysfunction induced by Ang II, suggesting that it could be an anticachectic agent that induces direct effects on cardiac muscles.

Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

The Potential Pharmacological Effects of Natural Product Withaferin A in Cancer: Opportunities and Challenges for Clinical Translation.

Cancer is one of the biggest health concerns with a complex pathophysiology. Currently, available chemotherapeutic drugs are showing deleterious side effects, and tumors often show resistance to treatment. Hence, extensive research is required to develop new treatment strategies to fight against cancer. Natural resources from plants are at the forefront of hunting novel drugs to treat various types of cancers. Withaferin A (WA) is a naturally occurring withanolide, a biologically active component obtained from the plant Ashwagandha. Various in vitro and in vivo oncological studies have reported that Withaferin A (WA) has shown protection from cancer. WA shows its activity by inhibiting the growth and proliferation of malignant cells, apoptosis, and inhibiting angiogenesis, metastasis, and cancer stem cells (CSCs). In addition, WA also showed chemo- and radio-sensitizing properties. Besides the beneficiary pharmacological activities of WA, a few aspects like pharmacokinetic properties, safety, and toxicity studies are still lacking, hindering this potent natural product from entering clinical development. In this review, we have summarized the various pharmacological mechanisms shown by WA in in vitro and in vivo cancer studies and the challenges that must be overcome for this potential natural product's clinical translation to be effective.

Novel Withanolides from Tubocapsicum anomalum Suppress Triple-Negative Breast Cancer by Triggering Apoptosis and p53-ASCT2-SLC7A11-Mediated Ferroptosis.

Triple-negative breast cancer (TNBC) is a malignant breast cancer. There is an urgent need for effective drugs to be developed for TNBC. Tubocapsicum anomalum (T. anomalum) has been reported to have an anti-tumor effect, and six novel withanolides were isolated from it and designated as TAMEWs. However, its anti-TNBC effect is still unknown. The results of an MTT assay indicated a higher sensitivity of TNBC cells to TAMEWs compared to other cells. TAMEWs induced apoptosis via mitochondrial dysfunction. They caused increased levels of lipid ROS and Fe2+, with downregulation of GSH and cystine uptake, and it has been confirmed that TAMEWs induced ferroptosis. Additionally, the results of Western blotting indicate that TAMEWs significantly decrease the expressions of ferroptosis-related proteins. Through further investigation, it was found that the knockdown of the p53 gene resulted in a significant reversal of ferroptosis and the expressions of its associated proteins SLC7A11, ASCT2, and GPX4. In vivo, TAMEWs suppressed TNBC growth with no obvious damage. The IHC results also showed that TAMEWs induced apoptosis and ferroptosis in vivo. Our findings provide the first evidence that TAMEWs suppress TNBC growth through apoptosis and ferroptosis.

Combined untargeted metabolomics and network pharmacology approaches to reveal the therapeutic role of withanolide B in psoriasis.

Psoriasis is a refractory inflammatory skin disorder in which keratinocyte hyperproliferation is a crucial pathogenic factor. Up to now, it is commonly acknowledged that psoriasis has a tight connection with metabolic disorders. Withanolides from Datura metel L. (DML) have been proved to possess anti-inflammatory and anti-proliferative properties in multiple diseases including psoriasis. Withanolide B (WB) is one of the abundant molecular components in DML. However, existing experimental studies regarding the potential effects and mechanisms of WB on psoriasis still remain lacking. Present study aimed to integrate network pharmacology and untargeted metabolomics strategies to investigate the therapeutic effects and mechanisms of WB on metabolic disorders in psoriasis. In our study, we observed that WB might effectively improve the symptoms of psoriasis and alleviate the epidermal hyperplasia in imiquimod (IMQ)-induced psoriasis-like mice. Both network pharmacology and untargeted metabolomics results suggested that arachidonic acid metabolism and arginine and proline metabolism pathways were linked to the treatment of psoriasis with WB. Meanwhile, we also found that WB may affect the expression of regulated enzymes 5-lipoxygenase (5-LOX), 12-LOX, ornithine decarboxylase 1 (ODC1) and arginase 1 (ARG1) in the arachidonic acid metabolism and arginine and proline metabolism pathways. In summary, this paper showed the potential metabolic mechanisms of WB against psoriasis and suggested that WB would have greater potential in psoriasis treatment.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. METHODS: Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. RESULTS: Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. CONCLUSION: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

Withaferin A-Encapsulated PEGylated Nanoliposomes Induce Apoptosis in B16F10 Melanoma Cells by Regulating Bcl2 and Bcl xl Genes and Mitigates Murine Solid Tumor Development.

Withaferin A (WA) is a natural steroidal lactone with promising pharmacological activities, but its poor solubility and bioavailability hinder its clinical application. The liposomal drug delivery system has attracted considerable attention to overcome the delivery limitations of pharmacological agents. The present study investigated the effect of WA-loaded pegylated nanoliposomes (LWA) on in vitro and in vivo B16F10 melanoma tumor models. In vitro results showed that LWA had significantly (P < 0.01) higher cytotoxicity than free WA and induced ROS-mediated apoptosis in B16F10 cells. Transwell cell migration and invasion studies demonstrated that LWA treatment significantly (P < 0.01) decreased the migratory and invasive capacities of melanoma cells compared with WA. In vivo study revealed that treatment significantly (P < 0.01) reduced tumor growth in experimental animals compared with WA or tumor control. Also, LWA administration remarkably inhibited tumor cell proliferation by downregulating the expression of Ki-67 and Cyclin D1 and induced apoptosis by regulating the expression of Bax, Bcl2, and Bcl xl levels. Our results strongly suggest that LWA could be a promising therapeutic formulation for treating malignant melanoma.

Withaferin A alters the expression of microRNAs 146a-5p and 34a-5p and associated hub genes in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. Due to the absence of obvious therapeutic targets, microRNAs (miRNAs) provide possible hope to treat TNBC. Withaferin A (WA), a steroidal lactone, possesses potential anticancer activity with lesser side effects. The present study identifies hub genes (CDKN3, TRAF6, CCND1, JAK1, MET, AXIN2, JAG1, VEGFA, BRCA1, E2F3, WNT1, CDK6, KRAS, MYB, MYCN, TGFßR2, NOTCH1, SIRT1, MYCN, NOTCH2, WNT3A) from the list of predicted targets of the differentially expressed miRNAs (DEMs) in WA-treated MDA-MB-231 cells using in silico protein-protein interaction network analysis. CCND1, CDK6, and TRAF6 hub genes were predicted as targets of miR-34a-5p and miR-146a-5p, respectively. The study found the lower expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells, and further, it was observed that WA treatment effectively restored the lost expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells. An anti-correlation expression pattern was found among the miR-34a-5p and miR-146a-5p and the respective target hub genes in WA-treated TNBC cells. In conclusion, WA might exert anti-cancer effect in TNBC cells by inducing miR-34a-5p and miR-146a-5p expressions and decreasing CCND1, CDK6, and TARF6 target hub genes in TNBC cells.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.