Neem Leaf Glycoprotein disrupts exhausted CD8+ T cell-mediated cancer stem cell aggression.

Targeting exhausted CD8+T cell (TEX) induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune-dependent tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify TEX to target CSCs successfully. Here we report that NLGP's therapeutic administration significantly reduced TEX -associated CSC virulence in in vivo B16-F10 melanoma tumor model. Similar trend was observed in in vitro generated TEX and B16-F10/MCF7 co-culture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, SOX2 expression. Cell cycle analysis revealed that NLGP-educated TEX efficiently pushed CSCs out of quiescent-phase (G0G1) into synthesis-phase (S), supported by hyper-phosphorylation of G0G1-S transitory cyclins and Rb-proteins. This rendered quiescent CSCs susceptible to s-phase targeting chemotherapeutic drugs like 5-Fluorouracil (5FU). Consequently combinatorial treatment of NLGP and 5FU brought optimal CSC targeting efficiency with increase in apoptotic bodies and pro-apoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU associated toxicity. Furthermore, Dectin-1 mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis was deciphered as the involved signalling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug-target to counter T cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8+T cell induced CSC aggravation. Implications: Our study recommends that NLGP-immunotherapy can be utilized to counter ramifications of T cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.

Cancer stem cell-immune cell crosstalk in breast tumor microenvironment: a determinant of therapeutic facet.

Breast cancer (BC) is globally one of the leading killers among women. Within a breast tumor, a minor population of transformed cells accountable for drug resistance, survival, and metastasis is known as breast cancer stem cells (BCSCs). Several experimental lines of evidence have indicated that BCSCs influence the functionality of immune cells. They evade immune surveillance by altering the characteristics of immune cells and modulate the tumor landscape to an immune-suppressive type. They are proficient in switching from a quiescent phase (slowly cycling) to an actively proliferating phenotype with a high degree of plasticity. This review confers the relevance and impact of crosstalk between immune cells and BCSCs as a fate determinant for BC prognosis. It also focuses on current strategies for targeting these aberrant BCSCs that could open avenues for the treatment of breast carcinoma.

Regulation of RNA methylation by therapy treatment, promotes tumor survival.

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

Cetuximab-conjugated PLGA nanoparticles as a prospective targeting therapeutics for non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most prevalent cancers diagnosed worldwide, yet managing it is still challenging. The epidermal growth factor receptor (EGFR) exhibits aberrant signalling in a wide range of human cancers, and it is reported to overexpress in most NSCLC cases. The monoclonal antibody [Cetuximab (Cet)] was conjugated onto the surface of the poly (lactide-co-glycolide) (PLGA) nanoparticles which were loaded with docetaxel (DTX) for the development of targeted therapy against lung cancer. This site-specific delivery system exhibited an enhanced cellular uptake in lung cancer cells which overexpress EGFR (A549 and NCI-H23). The nanoparticles also showed better therapeutic effectiveness against NSCLC cells, as evidenced by reduced IC50 values, cell cycle arrest at the G2/M phase, and increased apoptosis. The improved efficacy and in vivo tolerance of Cet-DTX NPs were demonstrated in benzo(a)pyrene (BaP)-induced lung cancer mice model. Histopathological analysis showed that intravenous injection of Cet-DTX NP to mice carrying lung cancer greatly reduced tumour development and proliferation. Comparing Cet-DTX NP to free drug and unconjugated nanoparticles, it also had negligible side effects and improved survival rates. Therefore, Cet-DTX NPs present a promising active targeting carrier for lung tumour-NSCLC-selective treatment.

Tumor activated platelets induce vascular mimicry in mesenchymal stem cells and aid metastasis.

Extent of metastasis influences activation of platelets in tumor-microenvironment. Activated platelets potentiate mesenchymal-stem-cells (MSCs) to migrate in secondary metastatic sites without participation in process of invasion. Presence of higher percentage of MSCs along with activated-platelets induces formation of vascular-mimicry (VM). The pathophysiology, VM, has already been reported in multiple types of cancer including lung, ovary, melanoma etc. and related to poor-prognosis. Interaction of MSCs with platelets in cell-to-cell contact dependent manner is essential for their migration, thereby, VM. Evidences are obtained suggesting that under influence of tumor-associated-activated-platelets, expressions of vimentin, ve-cadherin are increased, along with decrease in e-cadherin on CD105+ MSCs in both mRNA and protein levels that may help in formation of vessel like structure in VM. Adoptive transfer of MSCs along with tumor-activated-platelets causes greater B16 melanoma metastasis at lungs in comparison to MSCs with non-activated platelets. Presence of CD105+Vimentin+ MSCs in vessel like structure in the metastatic lung confirms the involvement of platelet-activated-MSCs in VM, thereby, in metastasis.

Deregulation of the CD44-NANOG-MDR1 associated chemoresistance pathways of breast cancer stem cells potentiates the anti-cancer effect of Kaempferol in synergism with Verapamil.

Chemoresistance is an imminent therapeutic challenge for breast cancer. Previous evidence suggests that breast cancer stem cells (BCSC) develop resistance through upregulation of stemness and chemo-evasion markers viz. SOX2, OCT4, NANOG, MDR1 and CD44, following anticancer chemotherapeutic treatments. Early studies suggest an inhibitory role of Kaempferol in BCSC propagation through downregulation of epithelial to mesenchymal transition. We hypothesized that the pathway involved in chemoresistance could be effectively addressed through Kaempferol (K), alone or in combination with Verapamil (V), which is an inhibitor of MDR1. We used K in combination with V, in multiple assays to determine if there was an inhibitory effect on BCSC. Both K and KV attenuated pH-dependent mammosphere formation in primary BCSC and MDA-MB-231 cells. RNA and protein (immunocytochemistry, western blot) expression of candidate markers viz. SOX2, OCT4, NANOG, MDR1 and CD44 were carried out in the presence or absence of candidate drugs in ex-vivo grown primary BCSC and MDA-MB-231 cell line. Immunoprecipitation assay, cell cycle analysis was carried out in MDA-MB-231. Our candidate drugs were not only anti-proliferative, but also downregulated candidate genes expression at RNA and protein level in both settings, with more robust efficacy in KV treatment than K; induced G2/M dependent cell cycle arrest, and interrupted physical association of CD44 with NANOG as well as MDR1 in MDA-MB-231. In primary tumor explant but not in adjacent normal tissue, our candidate drugs K and KV induced robust γH2AX expression. Thus, our candidate drugs are effective in attenuating BCSC survival.

Eugenol emerges as an elixir by targeting ß-catenin, the central cancer stem cell regulator in lung carcinogenesis: an in vivo and in vitro rationale.

According to population-based studies, lung cancer has become one of the leading causes of death globally in males and is also rising in females at an alarming rate. The aim of this study was to exploit the inherent properties of eugenol to restrict the growth of cancer cells in a tobacco-related human carcinogen NDEA-induced lung carcinogenesis model in vivo as a chemopreventive agent. More precisely, by utilizing its abundance in nature, eugenol (a component of clove) was utilized to establish the molecular mechanism of chemoprevention in the NDEA-induced mouse lung carcinogenesis model in a substantial cost-effective manner and was validated in the A549 human lung cancer cell line. Our study especially targeted the tiny, drug-resistant, and most virulent subpopulation of cancer cells called CSCs by targeting their regulator molecule ß-catenin. The non-toxic dosage of eugenol was shown to enhance apoptosis, simultaneously suppressing cell proliferation in the lung tissue of carcinogen-treated mice without affecting the normal mice. Combining cellular apoptosis and proliferation, eugenol showed an exceptional chemopreventive potential in this lung carcinogenesis model. Importantly, eugenol strongly restricted the lung carcinoma in the mild dysplastic stage as a chemopreventive agent. The molecular analysis remarkably depicted the restriction of ß-catenin nuclear transportation. The minimized total ß-catenin pool and induced N-terminal Ser37 phosphorylation form after eugenol treatment resulted in its cytoplasmic degradation. Consequently, CSC markers such as CD44, Oct4, EpCAM, and Notcht1, whose expression is dependent on ß-catenin decreased significantly, as proven by IHC, ICC, and WB analysis both in vivo and in vitro. The in vitro secondary sphere formation assay also proved the remarkably repressed CSC population, and hence the virulence. In another way, eugenol was proven to significantly enhance the degradation of ß-catenin when treated with the CK1α inhibitor D4476 in vitro by Western blot. CK1α in the Wnt/ß-catenin pathway plays a crucial role for tagging with the N-terminal Ser45 phosphorylation of ß-catenin, which ultimately opens a position for the decisive phosphorylation by GSK3ß at the Ser37 residue to take place. Thus, the conclusive extermination of CSCs achieved that was associated with recurrence due to treatment failure. That can help to achieve a longer and better quality of life in a natural, economical way.

Anti-Metastatic Potential of a Novel Xanthone Sourced by Swertia chirata Against In Vivo and In Vitro Breast Adenocarcinoma Frameworks.

BACKGROUND: The Anticancer property of Swertia chirata has been well established. It forms a rich source of compounds to which its anticancer property can be attributed, among the compounds found in S. chirata xanthones form an important group. Among the most abundant xanthones found in S. chirata, 1,5,8-trihydroxy-3-methoxy xanthone (TMX) was found to be most effective. As metastasis is the underlying cause of most cancer-related deaths, in this study, we evaluated the anti-metastatic potential of TMX against adenocarcinoma both in vivo and in vitro. MATERIALS AND METHODS: In vivo anti-metastatic potential was proved by histological evidence of different organs, giemsa staining of bone marrow, subcutaneous re-injection of the aberrant bone marrow cells into the right flank of the mice to observe the formation of tumors and analyzing the markers related to metastasis by immunohistochemistry (IHC) and western blot. In vitro validation of anti-metastatic potential was carried out against human breast adenocarcinoma cell line MCF-7 by primarily analyzing the migratory property of cells through scratch wound healing assay and the ability of cells to form colonies. The re-validation part was performed by western blot of markers related to metastasis and real-time analysis of EMT related markers. RESULTS: In vivo, TMX treatment restricted metastasis of EAC induced solid tumor to liver, lung, bone marrow, and validation of this finding was achieved by down regulation of metastatic and EMT markers.  In vitro, TMX treatment restricted migratory and colony forming ability of MCF-7 cells by down regulating metastatic and EMT markers. CONCLUSION: It was proved from our study that TMX treatment successfully reduced the metastatic potential of EAC induced solid tumor, with in vitro validation TMX on the MCF-7 cell line.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives: Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231). Methods: Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting. Results: Among all the xanthones tested XD showed IC50at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines. Interpretation & conclusions: Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Eugenol restricts Cancer Stem Cell population by degradation of ß-catenin via N-terminal Ser37 phosphorylation-an in vivo and in vitro experimental evaluation.

Eugenol a phenylpropanoid, predominantly found in clove is a very common spice in daily cuisine. It already reported to have anti-breast cancer activity. In this study, the effect of eugenol on CSC (Cancer Stem Cell) markers and its main regulator ß-catenin both in vivo Ehrlich Ascites Carcinoma (EAC) cell line and in vitro MCF-7 cell line was investigated with that of the untreated group. The therapeutic doses were found to significantly induce apoptosis leaving normal mice and cells unaffected. The in-depth analysis revealed the downregulation of ß-catenin thereby facilitating its degradation by N-terminal phosphorylation of Ser37 residue. Significant downregulation of various CSC markers was also observed in vivo after eugenol treatment those are regulated by the intracellular status of ß-catenin. These findings were validated by the effect of eugenol on the formation of the secondary sphere in vitro. Notable downregulation of the enriched stemness of secondary mammosphere was detected by the significantly decreased percentage of CD44+/CD24-/low population after eugenol treatment along with their distorted morphology and smaller the number of spheres. The underlying mechanism revealed significant downregulation of ß-catenin and the set of CSC markers along with their reduced mRNA expression in secondary sphere culture. Therefore, it can be concluded from the study that eugenol exerts its chemotherapeutic potential by impeding ß-catenin nuclear translocation thereby promoting its cytoplasmic degradation as a result stemness is being suppressed potentially even if in the enriched state. Therefore the study contributes to reduce the cancer-induced complications associated with the CSC population. This will ultimately confer the longer and improved patient's life.

Chemotherapeutic potential of novel non-toxic nucleoside analogues on EAC ascitic tumour cells.

Therapeutic efficacy of nucleoside analogues (NAs) like Gemcitabine, 5-fluorouracil in cancer treatment is already well established. Most of the known NAs are highly toxic to normal cells due to its non-specific action; thus searching for non-toxic NAs are still going on. For that purpose we have synthesised nine different NAs by alteration of their structural and functional groups. The aim of present study is to investigate the therapeutic potential of NAs against mice bearing breast adenocarcinoma cells at IC50 dose for 10 days treatment schedule. Results of the present study showed that, among the seven nucleoside analogues, NA-7 and NA-9 showed maximum therapeutic efficacy in controlling cancer cells by inhibiting cell proliferation and inducing apoptosis without any adverse effects to normal host cells. Additionally, NAs significantly decreased the tumour burden and enhanced survivability of host through generation of reactive oxygen species in tumour cells. These ultimately led to DNA damage, depolarisation of mitochondrial membrane potential and apoptosis in tumour cells. To find out the molecular mechanisms, we showed that administration of NA-7 and NA-9, down- regulating the expression of Bcl-2, cyclin D1, C-myc, P-21 and up-regulating the expression of P-53, Cyt-c, Bax, caspase-3 and caspase-9. The results suggest that NA-7 and NA-9 exhibits significant antitumor activity than 5-fluorouracil by modulating the cell cycle checkpoints and inducing apoptosis in Ehrlich ascites carcinoma (EAC)-bearing mice. Additionally, NA-7 and NA-9 did not show any clastogenic effect on bone marrow cells at sub-lethal dose. Thus, the present study clearly suggested therapeutic benefit of NAs by augmenting anticancer efficacy and diminishing toxicity to the host.

Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy.

KRAS, a frequently mutated G-quadruplex forming proto-oncogene is responsible for almost every type of cancer which can form a parallel G-quadruplex structure in the promoter region. G-quadruplex structure is one of the most important drug targets for modern cancer therapy for their unique structure and specificity. Here, we have screened several synthetic porphyrin-based compounds as potential KRAS G-quadruplex stabilizing ligands, using molecular modeling and docking studies. Two novel porphyrins: Porphyrin-1(Cobalt containing) and Porphyrin-2 (Palladium containing) evidenced high affinity towards KRAS-promoter/G-quadruplex. As KRAS mutation is prevalent in pancreatic cancer, the efficacy of these ligands against human pancreatic ductal carcinoma cell line PANC-1 and MiaPaCa2 were examined. Both the Porphyrins exhibited significant cytotoxicity and block metastasis by inhibiting Epithelial to messenchymal transition. In vivo studies confirmed both porphyrin compounds to be effective against EAC tumors along with significantly low toxicity against normal Swiss albino mice. The expression of KRAS gene in porphyrin-treated PANC-1, MiaPaCa2 and tumor-derived EAC cells were drastically reduced at both protein and RNA levels. Thus interaction of porphyrin-based ligands with G-quadruplex DNA at the promoter region of KRAS, might be utilized as a target for anticancer therapeutic strategy.

A Novel Approach of Synthesizing and Evaluating the Anticancer Potential of Silver Oxide Nanoparticles in vitro.

BACKGROUND: Development of novel strategies to kill cancer by sparing normal cells is of utmost importance. Apart from their known antimicrobial activity, only limited information has been recorded regarding the antitumor potential of biocompatible silver oxide nanoparticles (AgONPs). There is a need to evaluate the anticancer potential of biocompatible AgONPs in vitro. METHODS: A new approach of utilizing the leaf extract of Excoecaria agallocha was used to synthesize AgONPs. This was then characterized by ultraviolet-visible spectrophotometry, nanoparticle-tracking analysis, and ζ-potential analysis. Cytotoxicity and apoptotic potential were evaluated with an MTT assay and an annexin V-binding assay against the murine melanoma (B16F10), murine colon cancer (CT26), murine lung adenocarcinoma (3LL), and murine Ehrlich ascites carcinoma (EAC) cell lines. Cellular localization of AgONPs was evaluated on fluorescence microscopy. RESULTS: UV peaks at 270 and 330 nm indicated the formation of nanoparticles (NPs) and the NP-tracking analyzer revealed them to have a size of 228 nm. AgONPs exerted initial cytotoxicity, specifically against all the experimental malignant cells by sparing the normal cell lines. Moreover, AgONPs exert apoptosis equally on all the malignant cells in vitro and ex vivo. This cytotoxicity possibly occurs via the nuclear translocation of AgONPs as analyzed in B16F10 cells. CONCLUSIONS: AgONPs utilizing natural sources would be a new medicinal approach against a broad spectrum of malignancy.