Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection.

Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1ß treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.

Inflammation and Stem Cell Stochasticity of HPV-induced Cervical Cancer: Epigenetics Based Biomarkers through Microbiome and Metabolome for Personalized Medicine: A Systematic Review.

BACKGROUND: Chemoresistance by stemness in HPV-induced cervical carcinogenesis has significant implications for the overall disease-specific survival of the patients. To date, there are no reports related to the implications of significant aspects of inflammation and microbiome-- mediated epigenetics in cervical cancers. OBJECTIVE: The current systematic review delineates the significant aspects of the inflammation-related pathophysiology, cervical cancer diagnosis based on the HPV-indued stemness, and microbiome- mediated epigenetic markers to develop personalized therapies to target the stemness-acquired indefinitely dividing cancer stem cells. METHODS: We performed a systematic review without a meta- analysis. We searched several public databases, such as Pubmed, ReleMed, National Library of Medicine, and Scopus, related to inflammation, metabolomics, microbiome-mediated epigenetic markers, and HPV-induced stemness. RESULTS AND CONCLUSION: The review significantly described the correlation between microbial inflammation and stem cell stochasticity of HPV-Induced cervical cancer and the expression of epigenetics- based biomarkers through microbiome and metabolome to foster the cervical cancer progression. These are major risk factors that can cause cervical dysplasia with substantial therapy resistance in cervical cancer patients. The qualitative and quantitative examination of the spatial transcriptomic expression of these stemness markers in the dividing cervical cancer stem cells has significant implications in the clinical sector to develop early personalized medicine to prevent cervical precancerous lesions depending on the prognosis of the cervical cancer patients. Mainly, the combinatorial regimen of current therapeutic modalities, along with microbiome-related therapies with future landscape of epigenetics-modulated therapies, may enhance overall disease-specific survival by modulating the stochastic dynamics of basal epithelial cells across the cervical region.

Combinatorial Implications of Nrf2 Inhibitors with FN3K Inhibitor: In vitro Breast Cancer Study.

BACKGROUND: Platinum derivatives are chemotherapeutic agents preferred for the treatment of cancers including breast cancer. Oxaliplatin is an anticancer drug that is in phase II studies to treat metastatic breast cancer. However, its usage is constrained by chemoresistance and dose-related side effects. OBJECTIVE: The objective of this study is to examine the combinatorial efficacy of brusatol, an Nrf2 blocker, with oxaliplatin (a proven FN3K blocker in our study) in mitigating breast cancer growth in vitro. METHODS: We performed cytotoxicity assays, combination index (CI) analysis, colony formation assays, apoptosis assays, and Western blotting. RESULTS: Results of our study described the chemosensitizing efficacy of brusatol in combination with lowdose oxaliplatin against breast cancer through synergistic effects in both BT-474 and T47D cells. A significant mitigation in the migration rate of these cancer cells was observed with the combination regimen, which is equivalent to the IC-50 dose of oxaliplatin (125 µM). Furthermore, ROS-mediated and apoptotic modes of cell death were observed with a combinatorial regimen. Colony formation of breast cancer cell lines was mitigated with a combinatorial regimen of bursatol and oxaliplatin than the individual treatment regimen. FN3K expression downregulated with oxaliplatin in T47D cells. The mitigation of FN3K protein expression with a combination regimen was not observed but the Nrf2 downstream antioxidant signaling proteins were significantly downregulated with a combination regimen similar to individual drug regimens. CONCLUSION: Our study concluded the combination efficacy of phytochemicals like brusatol in combination with low-dose oxaliplatin (FN3K blocker), which could enhance the chemosensitizing effect in breast cancer and minimize the overall dose requirement of oxaliplatin.

Isoliensinine augments the therapeutic potential of paclitaxel in multidrug-resistant colon cancer stem cells and induced mitochondria-mediated cell death.

Previously we have reported the isoliensinine (ISO) potentates the therapeutic potential of cisplatin in cisplatin resistant colorectal cancer stem cells. The present study evaluates the chemo-sensitizing potential of the combinatorial regimen of ISO and Paclitaxcel (PTX) on multidrug-resistant (MDR)-HCT-15 cells to reduce the dose requirement of both ISO and PTX. The results of the present study suggest that treatment with the combinatorial regimen of ISO and PTX enhanced the cytotoxic effect with resultant increase in apoptosis in MDR-HCT-15 cells as evident from the altered cellular morphology, G2/M cell cycle arrest, propidium iodide uptake, Annexin V, increased intracellular Ca2+ accumulation, decreased mitochondrial membrane potential, diminished ATP production, PARP-1 cleavage, altered expression of ERK1/2, and apoptotic proteins. Treatment with combinatorial regimen of ISO and PTX also modulated the expression of the transcription factors SOX2, OCT4 which determine the stemness of cancer cells. Thus, results of the present study suggest that ISO and PTX combination regimen induces apoptosis in MDR-HCT-15 in a synergistic manner.

Impediment of Cancer by Dietary Plant-derived Alkaloids Through Oxidative Stress: Implications of PI3K/AKT Pathway in Apoptosis, Autophagy, and Ferroptosis.

The adverse toxicities and stemness are two major factors that constrained the usage of therapeutic strategies to target several cancer types. Previous studies explored the efficacy of PI3K/mTOR inhibitors, pan-PI3K inhibitors, and isoform-specific inhibitors against several cancer types, and many of them are currently in clinical trials. The current review described the efficacy of alkaloids derived from dietary plant sources in developing a new anti-cancer to reduce the prevalence of cancer through the modulation of apoptosis, autophagy, and ferroptosis. We have substantially collected the information pertinent to several intracellular pathways, including PI3K signaling, apoptosis, ferroptosis, and autophagy in modulating cancer progression mediated by the plantderived alkaloids such as daurisoline, dauricine, vasicine, vasicinone, 2-Acetyl-benzylamine, nuciferine, liensinine, gramine, and berbamine. These alkaloids exhibit significant anti-cancer potential to inhibit cancer cells by enhancing the intracellular ROS level and modulation of several signaling pathways, mainly through the PI3K/AKT pathway. These alkaloids can modulate chemotherapeutic agents' efficacy in various cancer cells, both in vitro and in vivo models. Overall the futures for the continued use of alkaloids from natural sources against cancer have to be extended, with the implementation of significant enhancements in the chemistry of these alkaloids for targeted delivery. In this review, we have selected major bioactive alkaloids of dietary and medicinal plants origin and discussed the anti-cancer and combinatorial therapeutic implications of these compounds with several FDA-approved drugs against various cancer cells.

Perspectives on the nanocarriers with miRNAs for targeting melanoma stemness through epigenetic regulation.

Several research reports delineated the significant role of miRNAs in cancer proliferation, and their modulatory role in cancer mitigation, and drug resistance. Melanoma cells have been acquiring stemness to several chemotherapeutic agents through drug efflux proteins, epigenetic modulation, and DNA repair. miRNAs could be applied as novel therapeutic modalities for treating several kinds of cancers to modulate these mechanisms involved in stemness. Nanocarriers to carry these tumor-targeting miRNAs to modulate stemness are a prominent strategy to overcome their low penetrability, minimal stability, and nonspecificity. We have searched several public databases such as PubMed, Medline, Google scholar, and NLM and obtained the information pertinent to the miRNA-based nanocarrier systems to target stemness through epigenetic modulation in melanomas. This review delineates that various miRNAs can modulate the stemness in melanomas by specific intricate epigenetic signaling, and other cell-based signaling mechanisms. Specific nanocarrier formulations with specific miRNAs are optimal methods to deliver these miRNAs in order to achieve significant entrapment efficiency, loading efficiency, and stability. Furthermore, the combinatorial regimen of FDA-approved chemotherapeutic molecules with tumor-targeting miRNAs and chemotherapy combined with nanocarriers can efficiently deliver the utmost therapeutic window by targeting tumor matrix, invasion, metastasis, and angiogenesis in melanomas. Substantial research should focus on the clinical application of this gene therapy in melanomas using these low immunogenic, highly degradable, and biocompatible combinatorial nanotherapeutic regimens.

Fabrication of Multifunctional Drug Loaded Magnetic Phase Supported Calcium Phosphate Nanoparticle for Local Hyperthermia Combined Drug Delivery and Antibacterial Activity.

Magnetic calcium phosphate nanoparticles are biocompatible and have attracted much attention as biomaterials for bone tissue engineering and theranostic applications. In this study, we report the fabrication of a biocompatible magnetic nickel ferrite supported fluorapatite nanoparticle as a bone substitute material with hyperthermia potential using a facile wet precipitation approach. The composition and magnetic properties of the sample were analyzed using X-ray diffraction (XRD) and a vibrating sample magnetometer (VSM). The presence of both magnetic (NiFe2O4 and γ-Fe2O3) and fluorapatite phases was identified, and the sample exhibited ferromagnetic behavior with saturation magnetization and coercivity of 3.08 emu/g and 109 Oe, respectively. The fabricated sample achieved the hyperthermia temperature of ∼43 °C under tumor mimic conditions (neglecting Brownian relaxation) in 2.67 min, and the specific loss power (SLP) was estimated to be 898 W/g(Ni+Fe) which is sufficient to prompt irreversible cell apoptosis. Biocompatibility of the synthesized nanoparticle was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay with fibroblast NIH 3T3 and L929 cells. An in vitro drug release experiment was conducted at pH 5 (tumor mimic) and 7.4 (physiological), which revealed a release of 49.8% in the former and 11.6% in the latter pH for 11 days. The prepared sample showed antibacterial activity against S. aureus.

Perspectives of using microRNA-loaded nanocarriers for epigenetic reprogramming of drug resistant colorectal cancers.

Epigenetic regulation by microRNAs (miRs) demonstrated a promising therapeutic potential of these molecules to regulate genetic activity in different cancers, including colorectal cancers (CRCs). The RNA-based therapy does not change genetic codes in tumor cells but can silence oncogenes and/or reactivate inhibited tumor suppressor genes. In many cancers, specific miRs were shown to promote or stop tumor progression. Among confirmed and powerful epigenetic regulators of colon carcinogenesis and development of resistance are onco-miRs, which include let-7, miR-21, miR-22, miR-23a, miR-27a, miR-34, miR-92, miR-96, miR-125b, miR-135b, miR-182, miR-200c, miR-203, miR-221, miR-421, miR-451, and others. Moreover, various tumor-suppressor miRs (miR-15b-5b, miR-18a, miR-20b, miR-22, miR-96, miR-139-5p, miR-145, miR-149, miR-197, miR-199b, miR-203, miR-214, miR-218, miR-320, miR-375-3p, miR-409-3p, miR-450b-5p, miR-494, miR-577, miR-874, and others) were found silenced in drug-resistant CRCs. Re-expression of tumor suppressor miR is complicated by the chemical nature of miRs that are not long-lasting compounds and require protection from the enzymatic degradation. Several recent studies explored application of miRs using nanocarrier complexes. This study critically describes the most successfully tested nanoparticle complexes used for intracellular delivery of nuclear acids and miRs, including micelles, liposomes, inorganic and polymeric NPs, dendrimers, and aptamers. Nanocarriers shield incorporated miRs and improve the agent stability in circulation. Attachment of antibodies and/or specific peptide or ligands facilitates cell-targeted miR delivery. Addressing in vivo challenges, a broad spectrum of non-toxic materials has been tested and indicated reliable advantages of lipid-based (lipoplexes) and polymer-based liposomes. Recent cutting-edge developments indicated that lipid-based complexes with multiple cargo, including several miRs, are the most effective approach to eradicate drug-resistant tumors. Focusing on CRC-specific miRs, this review provides a guidance and insights towards the most promising direction to achieve dramatic reduction in tumor growth and metastasis using miR-nanocarrier complexes.

Repurposing the Antibacterial Activity of Etoposide─A Chemotherapeutic Drug in Combination with Eggshell-Derived Hydroxyapatite.

Drug repurposing has been gaining increasing interest recently due to the reduction in development cost and reduced development timelines. Here, we report the antibacterial activity of the anticancer drug etoposide investigated in combination with the eggshell-derived hydroxyapatite (EHA). Hydroxyapatite (HA) is a well-known bioactive material with enhanced osteoconductivity and possesses superior drug delivery properties. In the present work, we have synthesized etoposide-loaded EHA by the wet precipitation method. The physicochemical characterization of the samples confirmed the composition and amount of drug encapsulation. Screening for antibacterial activity confirmed the antibacterial effect of etoposide against Staphylococcus aureus. Biofilm formation test on pristine and etoposide-loaded samples showed the inhibition of biofilm formation on etoposide loading, which was further studied by confocal laser scanning microscopy (CLSM) and colony forming units (CFUs). It has been found that etoposide-loaded HA exhibited a sustained release of the drug upto 168 h. Analysis of the inhibition mechanism of etoposide against S. aureus revealed damage to the cell membrane and has been quantified using flow cytometry by the uptake of propidium iodide. Etoposide-loaded eggshell-derived HA (EHA-ET) exhibited excellent bioactivity and cytocompatibility against mouse fibroblast cells (L929) and supressed the growth of osteosarcoma cells (MG-63). Our studies reveal that the EHA-ET has a great potential for treating osteosarcoma and osteomyelitis.

Reversal of cisplatin resistance by neferine/isoliensinine and their combinatorial regimens with cisplatin-induced apoptosis in cisplatin-resistant colon cancer stem cells (CSCs).

Cisplatin chemotherapy to the colorectal cancer cells (CRCs) is accompanied by dose-limiting adverse effects along with the acquisition of drug resistance implicating low therapeutic outcomes. The present study is aimed to evaluate the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimen of neferine/isoliensinine with cisplatin against CSCs (cisplatin resistant colon stem cells). CSCs were developed using pulse exposure of cisplatin to parental HCT-15 cells. Neferine/isoliensinine or combinatorial regimens of Neferine/isoliensinine and cisplatin exhibited a stronger cytotoxic activity against CSCs compared to control. IC50 doses were found to be 6.5 µM for neferine, 12.5 µM for isoliensinine, and 120 µM for cisplatin respectively. Furthermore, the combinatorial regimen of a low dose of cisplatin (40 µM) with 4 µM neferine/8 µM isoliensinine induced cell death in a synergistic manner as described by isobologram. Neferine/isoliensinine could confer extensive intracellular reactive oxygen species generation in CSCs. Neferine/isoliensinine or combinatorial regimens dissipated mitochondrial membrane potential and enhanced intracellular [Ca2+ ]i, which were measured by spectroflurimetry. Furthermore, these combinatorial regimens induced a significant increase in the sub G0 phase of cell cycle arrest and PI uptake and alleviated the expression of ERCC1 in CSCs. Combinatorial regimens or neferine/isoliensinine treatments downregulated the cell survival protein expression (PI3K/pAkt/mTOR) and activated mitochondria-mediated apoptosis by upregulating Bax, cytochrome c, caspase-3, and PARP cleavage expression while downregulating the BCl-2 expression in CSCs. Our study confirms the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimens of neferine/isoliensinine with a low dose of cisplatin against CSCs.

Dietary Phytochemicals as a Potential Source for Targeting Cancer Stem Cells.

The tumor microenvironment is composed of various types of cells that lead to tumor heterogeneity. In the middle of these populations, cancer stem cells play a vital role in the initiation and progression of cancer cells and are capable of self-renewal and differentiation processes. These cancer stem cells are resistant to conventional therapy such as chemotherapy and radiotherapy. To eradicate the cancer stem cells in the tumor environment, various natural product has been found in recent years. In this review, we have selected some of the natural products based on anticancer potential including targeting cancer cells and cancer stem cells. Further, this review explains the molecular mechanism of action of these natural products in various cancer stem cells. Therefore, targeting a multi-drug resistant cancer stem cell by natural products is a novel method to reduce drug resistance and adverse effect during conventional therapy.

The Cytoprotective and Anti-cancer Potential of Bisbenzylisoquinoline Alkaloids from Nelumbo nucifera.

Natural product therapy has been gaining therapeutic importance against various diseases, including cancer. The failure of chemotherapy due to its associated adverse effects promoted adjunct therapy with natural products. Phytochemicals exert anti-carcinogenic activities through the regulation of various cell signaling pathways such as cell survival, inflammation, apoptosis, autophagy and metastasis. The 'small molecule-chemosensitizing agents' from plants induce apoptosis in drug-resistant and host-immune resistant cancer cells in in vitro as well as in vivo models. For example, alkaloids from Nelumbo nucifera, liensinine, isoliensinine and neferine exert the anticancer activity through enhanced ROS generation, activation of MAP kinases, followed by induction of autophagy and apoptotic cell death. Likewise, these alkaloids also exert their cytoprotective action against cerebrovascular stroke/ischemic stroke, diabetes, and chemotherapy-induced cytotoxicity. Therefore, the present review elucidates the pharmacological activities of these bisbenzylisoquinoline alkaloids which include the cytoprotective, anticancer and chemosensitizing abilities against various diseases such as cardiovascular diseases, neurological diseases and cancer.

Neferine and isoliensinine enhance 'intracellular uptake of cisplatin' and induce 'ROS-mediated apoptosis' in colorectal cancer cells - A comparative study.

Cisplatin (CDDP) is a potent platinum-based chemotherapeutic agent used to treat solid tumors including colorectal cancer via inducing cytotoxicity. CDDP usage is limited due to the chemoresistance and associated adverse effects. A combinatorial regimen of phytochemicals with anticancer activity along with approved anticancer drugs seems to be a hopeful strategy against cancer treatment. Lotus-derived compounds such as neferine and isoliensinine have proven significant chemosensitizing activity in different cancer cells. Present study aims to compare chemosensitizing activity/anticancer potential of neferine/isoliensinine in combinatorial regimen with CDDP. Results documented that neferine/isoliensinine with CDDP augmented 'intracellular uptake of cisplatin' consequently apoptosis in HCT-15 cells exemplified by 'apoptotic morphological changes', 'S phase cell cycle arrest', 'ROS mediated oxidative stress' with the concomitant escalation in intracellular calcium & dissipation of MMP and activation of MAPK/PI3K/AKT pathway'. Furthermore, isoliensinine combination with CDDP exclusively enhanced CDDP uptake and induced more ROS-mediated apoptosis compared to other treatment regimens. Combination regimens induced downregulation of Bcl2 and upregulation of cytochrome c, caspase 3, 9, PARP cleavage indicating apoptosis induction through the intrinsic pathway. Thus, the results of the present study suggest that CDDP combination with neferine/isoliensinine augments the anticancer potential of CDDP in an additive manner and decrease CDDP dose requirement.

Design and synthesis of novel pyrrolo[2,3-a]carbazoles: 7-Chloro-2-oxo-3a-(2'-oxo-2',3'-dihydro-1'H-indol-3'-yl)-2,3,3a,4,5,10-hexahydro-pyrrolo[3,2-a]carbazole-1-carbonitrile as an efficient anticancer agent.

Highly efficient poly functionalized pyrrolo[3,2-a]carbazoles via ring contraction through rearrangement and intramolecular Michael addition reaction using one pot multicomponent reaction (MCR) is reported for the first time. Free radical scavenging and anticancer activities were determined by DPPH and MTT assays respectively. Of these, compound 8d exhibited most potent activity against HCT-15 human colon cancer cell lines with an IC50 value of 9.9 µM and low toxicity toward normal human red blood cells. The morphological changes were visualized using scanning electron microscopy (SEM) technique, intracellular ROS generation measured by spectrofluorometer and gene expression levels of caspase-3, caspase-9 and Bcl-2 were determined using Semi quantitates PCR analysis for the target compound. Further, the structure activity relationships were also carried out. The results of the present study revealed that among pyrrolo[3,2-a]carbazole compounds, 7-chloro-2-oxo-3a-(2'-oxo-2',3'-dihydro-1'H-indol-3'-yl)-2,3,3a,4,5,10-hexahydro-pyrrolo[3,2-a]carbazole-1-carbonitrile could be exploited as an excellent anticancer agent against colon cancer cells.

Synthesis and anticancer activity of novel curcumin-quinolone hybrids.

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase.