Protective effect of diindolylmethane-enriched dietary cabbage against doxorubicin-induced cardiotoxicity in mice.

Chemotherapy with doxorubicin (Dox) can lead to cardiotoxic effects, presenting a major complication in cancer therapy. Diindolylmethane (DIM), derived from cruciferous vegetables like cabbage, exhibits numerous health benefits. However, its clinical application is limited because of low bioavailability and suboptimal natural concentrations in dietary sources. To address this limitation, we developed a processing methodology, specifically fermentation and boiling, to enhance DIM levels in cabbage. High-performance liquid chromatography (HPLC) analysis revealed a threefold DIM increase in fermented cabbage and a substantial ninefold increase in fermented-boiled cabbage compared to raw cabbage. To evaluate the clinical implications, we formulated a DIM-enriched diet and administered it to mice undergoing Dox treatment. Our in vivo results revealed that Dox treatment led to cardiotoxicity, manifested by changes in body and heart weight, increased mortality, and severe myocardial tissue degeneration. Dietary administration of the DIM-enriched diet enhanced antioxidant defenses and inhibited apoptosis in the cardiac tissue by interfering with mitoptosis and increasing antioxidant enzyme expression. Interestingly, we found that the DIM-enriched diet inhibited the nuclear translocation of NF-kB in cardiac tissue, thereby downregulating the expression of inflammatory mediators such as TNF-α and IL-6. Further, the DIM-enriched diet significantly reduced serum cardiac injury markers elevated by Dox treatment. These results suggest that the DIM-enriched cabbage diet can serve as a complementary dietary intervention for cancer patients undergoing chemotherapy. Further, our research highlights the role of plant-based diets in reducing treatment side effects and improving the quality of life for cancer patients.

Dietary Diindolylmethane Enhances the Therapeutic Effect of Centchroman in Breast Cancer by Inhibiting Neoangiogenesis.

Tumor angiogenesis is primarily regulated by vascular endothelial growth factor and its receptor (VEGF-VEGFR) communication, which is involved in cancer cell growth, progression, and metastasis. Diindolylmethane (DIM), a dietary bioactive from cruciferous vegetables, has been extensively studied in preclinical models for breast cancer prevention and treatment. Nevertheless, the possible role of DIM in the angiogenesis and metastasis regulations in triple-negative breast cancer (TNBC) remains elusive. Here, we investigated the potential anti-angiogenic and anti-metastatic role of DIM in combination with centchroman (CC). We observed that the oral administration of the DIM and CC combination suppressed primary tumor growth and tumor-associated vascularization in 4T1 tumors. Further, the DIM and CC combination exhibited a strong inhibitory effect on VEGF-induced angiogenesis in matrigel plugs. The mechanistic study demonstrated that DIM and CC could effectively downregulate VEGFA expression in tumor tissue and strongly interact with VEGFR2 to block its kinase activity. Interestingly, the DIM and CC combination also suppressed the lung metastasis of the highly metastatic 4T1 tumors through the downregulation of FAK/MMP9/2 signaling and reversal of epithelial-to-mesenchymal transition (EMT). Overall, these findings suggest that DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy for treating TNBC.

Diindolylmethane Promotes Metabolic Crisis and Enhances the Efficacy of Centchroman in Breast Cancer: A 1H NMR-Based Approach.

Diindolylmethane (DIM) is a key metabolite of indole-3-carbinol found in cruciferous vegetables such as broccoli, cauliflower, and cabbage. DIM has been known for its anti-cancerous activity through various mechanisms. Most cancer cells, including triple-negative breast cancer (TNBC), adapt distinct metabolic reprogramming for rapid growth and proliferation. Hence, targeting metabolic dysregulation may provide a favorable therapeutic condition for the treatment of TNBC. Earlier, we found that DIM increases the intracellular accumulation of Centchroman (CC), a potential anticancer agent, thereby enhancing the therapeutic potential of CC against breast cancer. However, the role of DIM in regulating TNBC cellular metabolism remains unknown. In the current study, we investigated the potential therapeutic interventions of DIM in TNBC and its metabolic reprogramming in enhancing the efficacy of CC. We found that DIM induced metabolic catastrophe in TNBC cells by regulating aerobic glycolysis and intermediate metabolism. Further, the DIM and CC combination significantly inhibited the TNBC tumor growth in the 4T1-syngeneic model. The inhibition of tumor growth was associated with the downregulation of key aerobic glycolysis mediators such as PKM2, GLUT1, and hypoxia-inducible factor 1α (HIF-1α). This is a first-of-a-kind investigation linking DIM with aerobic glycolysis regulation and enhancing the treatment efficacy of CC against TNBC. Therefore, these findings suggest that DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy for treating metabolically dysregulated TNBC.

Extract of Murraya koenigii selectively causes genomic instability by altering redox-status via targeting PI3K/AKT/Nrf2/caspase-3 signaling pathway in human non-small cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Dietary bioactives have been used as alternative therapeutics to overcome various adverse effects caused by chemotherapeutics. Curry leaves are a widely used culinary spice and different parts of this plant have been used in traditional medicines. Curry leaves are a rich source of multiple bioactives, especially polyphenols and alkaloids. Therefore, extraction processes play a key role in obtaining the optimum yield of bioactives and their efficacy. PURPOSE: We aim to select an extraction process that achieves the optimum yield of bioactives in curry leaves crude extract (CLCE) with minimum solvent usage and in a shorter time. Further, to investigate the anticancer properties of CLCE and its mechanism against lung cancer. METHODS: Different extraction processes were performed and analyzed polyphenol content. The bioactives and essential oils present in curry leaves were identified through LC-MS/MS and GC-MS analysis. The cytotoxicity of microwave-assisted CLCE (MA-CLCE) was investigated through MTT and colony-forming assays. The DNA damage was observed by comet assay. The apoptotic mechanisms of MA-CLCE were investigated by estimating ROS production, depolarization of mitochondrial membrane potential (MMP), and apoptotic proteins. The glutathione assay estimated the antioxidant potential of MA-CLCE in normal cells. RESULTS: Generally, conventional extraction methods require high temperatures, extra energy input, and time. Recently, green extraction processes are getting wider attention as alternative extraction methods. This study compared different extraction processes and found that the microwave-assisted extraction (MAE) method yields the highest polyphenols from curry leaves among other extraction processes with minimum processing. The MA-CLCE functions as an antioxidant under normal physiological conditions but pro-oxidant to cancer cells. MA-CLCE scavenges free radicals and enhances the intracellular GSH level in alveolar macrophages in situ. We found that MA-CLCE selectively inhibits cell proliferation and induces apoptosis in cancer cells by altering cellular redox status. MA-CLCE induces chromatin condensation and genotoxicity through ROS-induced depolarization of MMP. The depolarization of MMP causes the release of cytochrome c into the cytosol and activates the apoptotic pathway in lung cancer cells. However, pretreatment with ascorbic acid, an antioxidant, inhibits the MA-CLCE-induced apoptosis by reducing ROS production, which impedes mitochondrial membrane disruption, preventing BAX/BCL-2 expression alteration. Simultaneously, MA-CLCE downregulates the expression of survival signaling regulator PI3K/AKT, which modulates Nrf-2. MA-CLCE also diminishes intracellular antioxidant proficiency by suppressing Nrf-2 expression, followed by HO-1 expressions. CONCLUSION: Among several extraction methods, MA-CLCE is rich in several bioactives, especially polyphenols, alkaloids, and essential oils. Here, we reported for the first time that MA-CLCE functions as a pro-oxidant to lung cancer cells and acts as an antioxidant to normal cells by regulating different cellular programs and signaling pathways. Therefore, it can be further developed as a promising phytomedicine against lung cancer.

Progress and promises of epigenetic drugs and epigenetic diets in cancer prevention and therapy: A clinical update.

Epigenetic modifications are heritable yet reversible, essential for normal physiological functions and biological development. Aberrant epigenetic modifications, including DNA methylation, histone modification, and non-coding RNA (ncRNA)-mediated gene regulation play a crucial role in cancer progression. In cellular reprogramming, irregular epigenomic modulations alter cell signaling pathways and the expression of tumor suppressor genes and oncogenes, resulting in cancer growth and metastasis. Therefore, alteration of epigenetic-status in cancer cells can be used as a potential target for cancer therapy. Several synthetic epigenetic inhibitors (epi-drugs) and natural epigenetic modulatory bioactives (epi-diets) have been shown to have the potential to alter the aberrant epigenetic status and inhibit cancer progression. Further, the use of combinatorial approaches with epigenetic drugs and diets has brought promising outcomes in cancer prevention and therapy. In this article, we have summarized the epigenetic modulatory activities of epi-drugs, epi-diets, and their combination against various cancers. We have also compiled the preclinical and clinical status of these epigenetic modulators in different cancers.

Identification of hub genes associated with EMT-induced chemoresistance in breast cancer using integrated bioinformatics analysis.

Chemoresistance is one of the major challenges in the treatment of breast cancer. Recent evidence suggests that epithelial-to-mesenchymal transition (EMT) plays a critical role in not only metastasis but also in chemoresistance, hence causing tumor relapse. This study aimed to identify the hub genes associated with EMT and chemoresistance in breast cancer affecting patient/clinical survival. Commonly differentially expressed genes (DEGs) during EMT and chemoresistance in breast cancer cells were identified using publicly available datasets, GSE23655, GSE39359, GSE33146 and GSE76540. Hierarchical clustering analysis was utilized to determine the commonly DEGs expression pattern in chemoresistant (CR) breast cancer cells. GSEA revealed that EMT-related genes sets were enriched in the CR samples. Further, we found that EMT-induced breast cancer cells showed overexpression of drug efflux transporters along with resistance to chemotherapeutic drug. Pathway enrichment analysis revealed that the commonly DEGs were enriched in immunological pathways, early endosome, protein dimerization, and proteoglycans in cancer. Further, we identified eight hub genes from the protein-protein interaction (PPI) network. We validated the gene expression levels of the hub genes among TCGA breast cancer samples using UALCAN. Survival analysis for the hub genes was performed using KM plotter, which showed a worse relapse-free survival (RFS) of the hub genes among breast cancer patients. In conclusion, this study identified eight hub genes that play an important role in the pathways underlying EMT-induced chemoresistance in breast cancer and can be used as therapeutic targets after clinical validation.

Dietary Piperine Suppresses Obesity-Associated Breast Cancer Growth and Metastasis by Regulating the miR-181c-3p/PPARα Axis.

Adipocyte-derived leptin activates multiple oncogenic signaling, leading to breast cancer cell progression and metastasis. Hence, finding effective strategies to inhibit the oncogenic effects of leptin would provide a novel approach for disrupting obesity-associated breast cancer. In the current study, we explored the role of piperine, a major plant alkaloid from Piper nigrum (black pepper), against leptin-induced breast cancer. Piperine treatment significantly inhibited leptin-induced breast cancer cell proliferation, colony formation, migration, and invasion. We found that piperine downregulated the expression of PPARα, a predicted target of miR-181c-3p. Mechanistically, piperine potentiates miR-181c-3p-mediated anticancer potential in leptin-induced breast cancer cells. Interestingly, the knockdown of PPARα reduced the proliferative potential of leptin-induced breast cancer cells. Further, oral administration of piperine inhibited breast tumor growth in diet-induced obese mice, accompanied by the upregulation of miR-181c-3p and downregulation of PPARα expression. Together, piperine represents a potential candidate for further development as an anticancer agent for treating obesity-associated breast cancer.

miR-145-5p as a predictive biomarker for breast cancer stemness by computational clinical investigation.

BACKGROUND: Breast tumors enriched with breast cancer stem cells (BCSCs), play a crucial role in metastasis and tumor relapse. Hence, targeting BCSCs may lead to efficacious breast cancer therapy. BCSCs have a unique expression of stemness markers, including Nanog, POU5F1, SOX2, and CD44, which play a vital role in cancer stem cell properties. However, the regulation of microRNAs (miRNAs)-mediated cancer stem cell marker expressions is largely unclear. METHODS: MIENTURNET was used to predict miRNA-target interactions. miR-TV, UALCAN and GEPIA databases were used to analyze the expression of miR-145-5p and SOX2. Survival analysis was obtained by cBioportal, KM plotter and Breast Cancer Gene-Expression Miner. RNAComposer was used to perform miRNA-mRNA duplex prediction. In vitro mRNA and miRNA analysis was performed by qRT-PCR. RESULTS: It was observed that miR-145-5p was the common miRNA targeting stemness markers. miR-145-5p expression was found to be lower in breast cancer patients compared to healthy subjects. Based on survival analysis, low expression of miR-145-5p and high expression of SOX2 led to a poor overall survival rate in breast cancer patients. Pathway enrichment analysis indicated that SOX2 was highly enriched with transcription factors. Moreover, SOX2 expression level was also upregulated in axillary metastatic lymph nodules. Further, in vitro ectopic expression of miR-145-5p by its mimic downregulated the SOX2 expression compared to the control mimic. Overall, SOX2 was a direct target for miR-145-5p as per the binding and minimal-free energy. CONCLUSIONS: In this study, miR-145-5p targeting SOX2 was identified as a potential predictive biomarker for breast cancer stemness.

Dietary bioactive diindolylmethane enhances the therapeutic efficacy of centchroman in breast cancer cells by regulating ABCB1/P-gp efflux transporter.

Overexpression of drug efflux transporters is commonly associated with multidrug-resistance in cancer therapy. Here for the first time, we investigated the ability of diindolylmethane (DIM), a dietary bioactive rich in cruciferous vegetables, in enhancing the efficacy of Centchroman (CC) by modulating the drug efflux transporters in human breast cancer cells. CC is a selective estrogen receptor modulator, having promising therapeutic efficacy against breast cancer. The combination of DIM and CC synergistically inhibited cell proliferation and induced apoptosis in breast cancer cells. This novel combination has also hindered the stemness of human breast cancer cells. Molecular docking analysis revealed that DIM had shown a strong binding affinity with the substrate-binding sites of ABCB1 (P-gp) and ABCC1 (MRP1) drug-efflux transporters. DIM has increased the intracellular accumulation of Hoechst and Calcein, the substrates of P-gp and MRP1, respectively, in breast cancer cells. Further, DIM stimulates P-gp ATPase activity, which indicates that DIM binds at the substrate-binding domain of P-gp, and thereby inhibits its efflux activity. Intriguingly, DIM enhanced the intracellular concentration of CC by inhibiting the P-gp and MRP1 expression as well as activity. The intracellular retaining of CC has increased its efficacy against breast cancer. Overall, DIM, a dietary bioactive, enhances the anticancer efficiency of CC through modulation of drug efflux ABC-transporters in breast cancer cells. Therefore, DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy against human breast cancer.

Culinary spice bioactives as potential therapeutics against SARS-CoV-2: Computational investigation.

BACKGROUND: Coronavirus disease-2019 (COVID-19) is an infectious pandemic caused by SARS-CoV-2. SARS-CoV-2 main protease (Mpro) and spike protein are crucial for viral replication and transmission. Spike protein recognizes the human ACE2 receptor and transmits SARS-CoV-2 into the human body. Thus, Mpro, spike protein, and ACE2 receptor act as appropriate targets for the development of therapeutics against SARS-CoV-2. Spices are traditionally known to have anti-viral and immune-boosting activities. Therefore, we investigated the possible use of selected spice bioactives against the potential targets of SARS-CoV-2 using computational analysis. METHODS: Molecular docking analysis was performed to analyze the binding efficiency of spice bioactives against SARS-CoV-2 target proteins along with the standard drugs. Drug-likeness properties of selected spice bioactives were investigated using Lipinski's rule of five and the SWISSADME database. Pharmacological properties such as ADME/T, biological functions, and toxicity were analyzed using ADMETlab, PASS-prediction, and ProTox-II servers, respectively. RESULTS: Out of forty-six spice bioactives screened, six bioactives have shown relatively better binding energies than the standard drugs and have a higher binding affinity with at least more than two targets of SARS-CoV-2. The selected bioactives were analyzed for their binding similarities with the standard drug, remdesivir, towards the targets of SARS-CoV-2. Selected spice bioactives have shown potential drug-likeness properties, with higher GI absorption rate, lower toxicity with pleiotropic biological roles. CONCLUSIONS: Spice bioactives have the potential to bind with the specific targets involved in SARS-CoV-2 infection and transmission. Therefore, spice-based nutraceuticals can be developed for the prevention and treatment of COVID-19.

Centchroman prevents metastatic colonization of breast cancer cells and disrupts angiogenesis via inhibition of RAC1/PAK1/ß-catenin signaling axis.

AIMS: We have previously reported that Centchroman (CC), an oral contraceptive drug, inhibits breast cancer progression and metastasis. In this study, we investigated whether CC inhibits local invasion of tumor cells and/or their metastatic colonization with detailed underlying mechanisms. MAIN METHODS: The effect of CC on the experimental metastasis and spontaneous metastasis was demonstrated by using tail-vein and orthotopic 4T1-syngeneic mouse tumor models, respectively. The anti-angiogenic potential of CC was evaluated using well established in vitro and in vivo models. The role of RAC1/PAK1/ß-catenin signaling axis in the metastasis was investigated and validated using siRNA-mediated knockdown of PAK1 as well as by pharmacological PAK1-inhibitor. KEY FINDINGS: The oral administration of CC significantly suppressed the formation of metastatic lung nodules in the 4T1-syngeneic orthotopic as well as experimental metastatic models. More importantly, CC treatment suppressed the tube formation and migration capacities of human umbilical vein endothelial cells (HUVEC) and inhibited pre-existing vasculature as well as the formation of neovasculature. The suppression of migration and invasion capacities of metastatic breast cancer cells upon CC treatment was associated with the inhibition of small GTPases (Rac1 and Cdc42) concomitant with the downregulation of PAK1 and downstream ß-catenin signaling. In addition, CC upregulated the expression of miR-145, which is known to target PAK1. SIGNIFICANCE: This study warrants the repurposing of CC as a potential therapeutic agent against metastatic breast cancer.

Epigenetics of Breast Cancer: Clinical Status of Epi-drugs and Phytochemicals.

Epigenetics refers to alterations in gene expression due to differential histone modifications and DNA methylation at promoter sites of genes. Epigenetic alterations are reversible and are heritable during somatic cell division, but do not involve changes in nucleotide sequence. Epigenetic regulation plays a critical role in normal growth and embryonic development by controlling transcriptional activities of several genes. In last two decades, these modifications have been well recognized to be involved in tumor initiation and progression, which has motivated many investigators to incorporate this novel field in cancer drug development. Recently, growing number of epigenetic changes have been reported that are involved in the regulations of genes involved in breast tumor growth and metastasis. Drugs possessing epigenetic modulatory activities known as epi-drugs, mainly the inhibitors of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs). Some of these drugs are undergoing different clinical trials for breast cancer treatment. Several phytochemicals such as green tea polyphenols, curcumin, genistein, resveratrol and sulforaphane have also been shown to alter epigenetic modifications in multiple cancer types including breast cancer. In this chapter, we summarize the role of epigenetic changes in breast cancer progression and metastasis. We have also discussed about various epigenetic modulators possessing chemopreventive and therapeutic efficacy against breast cancer with future perspectives.

Epigenetics of skin cancer: Interventions by selected bioactive phytochemicals.

The prevalence and risk of skin cancer have been increasing over past three decades. Two major types of skin cancer observed in humans are melanoma and nonmelanoma. Nonmelanoma further subdivided into basal cell carcinoma and squamous cell carcinoma. Melanoma arises from melanocyte which locates at the bottom layer of skin epidermis, which primarily protects the skin from being exposed to external factors. Melanoma is less common among all other types of skin cancers but causes higher mortality. Epigenetic regulation associated with the transcriptional activation and inactivation of genes plays a major role in various disease progression including skin cancer. The major epigenetic changes observed at cellular level include DNA methylation, histone modifications, and miRNA-mediated gene regulation. The aberrant pattern in these epigenetic processes leads to altered expression of several genes involved in cell cycle, cell proliferation, cell motility, and apoptosis. Several natural bioactive phytochemicals have been shown to exhibit epigenetic modulatory capability and act as chemopreventive as well as therapeutic agents. In this review, we mainly discuss the major epigenetic modifications observed in melanoma and the epigenetic modulatory role of selected bioactive phytochemicals against the skin cancer.