Acidic pH is a crucial intrinsic property of the microenvironment of most solid tumors. Hence, the use of pH sensitive tumor targeting nanoparticles is an attractive approach to enhance the therapeutic efficacy of anti-cancer agents in solid tumors. Chitosan nanoparticles (CHNPs) have been widely explored in the area of cancer drug delivery; nevertheless their true potential as a pH responsive targeted drug delivery vehicle in cancer therapy has not been deciphered yet as most of the research is limited to pH dependent stability and drug release. In the present study, we investigate the direct effect of pH in synergy with RGD peptide based targeting on the therapeutic efficacy of chitosan nanoparticles (RGD-CHNPs) in breast cancer. Furthermore, for the first time we performed a comprehensive study showing the anti-tumor, anti-migratory and anti-angiogenic effect of raloxifene (Rlx) loaded CHNPs in breast cancer. We prepared stable formulations of raloxifene encapsulated CHNPs and RGD-CHNPs by the nontoxic ionic gelation method. pH dependent studies revealed that NPs possess higher stability and zeta potential along with enhanced cellular uptake at acidic pH (as present in solid tumors) compared to physiological pH. Furthermore, RGD conjugation enhanced the in vitro cellular uptake of CHNPs in α_vβ₃ integrin expressing breast cancer cells and induced higher cellular apoptosis in breast cancer cells which was further augmented by lower pH. Moreover, Rlx-RGD-CHNPs significantly inhibited breast cancer cell migration and angiogenesis. In vivo studies showed that Cy5.5 conjugated RGD-CHNPs can distinctly visualize tumors and Rlx-RGD-CHNPs significantly inhibit breast tumor growth without causing any toxic effect to normal tissue as confirmed by hematology and blood biochemical studies. Therefore, RGD-CHNPs could potentially enhance the therapeutic efficacy of chemotherapeutic drugs due to the synergistic effect of pH responsiveness and tumor specific targeting in breast cancer.

Melanoma is a form of cancer that initiates in melanocytes. Melanoma has multiple phenotypically distinct subpopulation of cells, some of them have embryonic like plasticity which are involved in self-renewal, tumor initiation, metastasis and progression and provide reservoir of therapeutically resistant cells. Cancer stem cells (CSCs) can be identified and characterized based on various unique cell surface and intracellular markers. CSCs exhibit different molecular pattern with respect to non-CSCs. They maintain their stemness and chemoresistant features through specific signaling cascades. CSCs are weak in immunogenicity and act as immunosupressor in the host system. Melanoma treatment becomes difficult and survival is greatly reduced when the patient develop metastasis. Standard conventional oncology treatments such as chemotherapy, radiotherapy and surgical resection are only responsible for shrinking the bulk of the tumor mass and tumor tends to relapse. Thus, targeting CSCs and their microenvironment niche addresses the alternative of traditional cancer therapy. Combined use of CSCs targeted and traditional therapies may kill the bulk tumor and CSCs and offer a promising therapeutic strategy for the management of melanoma.

Breast cancer is one of the most commonly diagnosed invasive cancers among women around the world. Among several subtypes, triple negative breast cancer (TNBC) is highly aggressive and chemoresistant. Treatment of TNBC patients has been challenging due to heterogeneity and devoid of well-defined molecular targets. Thus, identification of novel effective and selective agents against TNBC is essential. We used epoxyazadiradione to assess the cell viability, mitochondrial potential, ROS level, cell migration, apoptosis and protein expression in cell culture models of TNBC MDA-MB-231 and ER+ MCF-7 breast cancer cells. The molecular mechanism was examined in two different type of breast cancer cells in response to epoxyazadiradione. We have also analyzed the effect of epoxyazadiradione on breast tumor growth using in vivo mice model. In this study, we for the first time investigated that out of 10 major limonoids isolated from Azadirachta indica, epoxyazadiradione exhibits most potent anti-cancer activity in both TNBC and ER+ breast cancer cells. Epoxyazadiradione induces apoptosis and inhibits PI3K/Akt-mediated mitochondrial potential, cell viability, migration and angiogenesis. It also inhibits the expression of pro-angiogenic and pro-metastatic genes such as Cox2, OPN, VEGF and MMP-9 in these cells. Furthermore, epoxyazadiradione attenuates PI3K/Akt-mediated AP-1 activation. Our in vivo data revealed that epoxyazadiradione suppresses breast tumor growth and angiogenesis in orthotopic NOD/SCID mice model. Our findings demonstrate that epoxyazadiradione inhibits PI3K/Akt-dependent mitochondrial depolarisation, induces apoptosis and attenuates cell migration, angiogenesis and breast tumor growth suggesting that this compound may act as a potent therapeutic agent for the management of breast cancer.

Cancer stem cells (CSCs) are known to potentiate tumor initiation and maintenance in Oral Squamous Cell Carcinoma (OSCC). Increasing evidences suggest that CD44 ^high CD24 ^low population in OSCC are potential CSCs. MicroRNAs (miRNAs) have emerged as crucial players in tumor development. However, their role in maintenance of OSCC stem cells remains unclear. Here we report that CD44 ^high CD24 ^low population within OSCC cells and primary HNSCC tumors have an elevated expression of miR-146a. Moreover, over-expression of miR-146a results in enhanced stemness phenotype by augmenting CD44 ^high CD24 ^low population. We demonstrate that miR-146a induces stemness by stabilizing β-catenin with concomitant loss of E-cadherin and CD24. Interestingly, CD24 is identified as a novel functional target of miR-146a and ectopic expression of CD24 abrogates miR-146a driven potential CSC phenotype. Mechanistic analysis reveals that higher CD24 levels inhibit AKT phosphorylation leading to β-catenin degradation. Using stably expressing miR-146a/CD24 OSCC cell lines, we also validate that the miR-146a/ CD24/AKT loop significantly alters tumorigenic ability in vivo . Furthermore, we confirmed that β-catenin trans-activates miR-146a, thereby forming a positive feedback loop contributing to stem cell maintenance. Collectively, our study demonstrates that miR-146a regulate CSCs in OSCC through CD24-AKT-β-catenin axis. Highlights: MiR-146a induces cancer stem cell characteristics in OSCC by targeting CD24 CD24 abrogates miR-146a mediated stemness via β-catenin degradation in non-CSCs Akt/Wnt pathway is critical for sustenance of miR-146a driven potential CSCs The miR-146a/CD24/AKT loop significantly alters tumorigenic ability in vivo