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Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1α led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1α altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1α by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5γ2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1α/EphA2/Laminin-5γ2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5γ2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC. [ABSTRACT FROM AUTHOR]

Vasculogenic mimicry (VM), an endothelial cell–independent alternative mechanism of blood supply to the malignant tumour, has long been considered as an adverse prognostic factor in many cancers. The correlation of VM with laminin‐5γ2 and the assessment of their harmonized expression as an independent risk factor have not been elucidated yet in oral squamous cell carcinoma (OSCC). CD31/PAS staining stratified 116 clinically diagnosed OSCC specimens into VM+ and VM− cohorts. The expression pattern of laminin‐5γ2 and its upstream modulator MMP2 was evaluated by immunohistochemistry and Western blot. The Kaplan–Meier and Cox regression analyses were performed to assess the survival and prognostic implications. The presence of VM demonstrated a significant correlation with the expression of laminin‐5γ2 (p <.001) and MMP2 (p <.001). This pattern was mirrored by the significant upregulation of laminin‐5γ2 and MMP2 in VM+ cohorts compared with the VM− ones. Furthermore, co‐expression of VM and laminin‐5γ2 was significantly associated with tumour grade (p =.010), primary tumour size (p <.001), lymph node metastasis (p =.001) and TNM stages (p <.001) but not with patients' age, gender, tobacco and alcohol consumption habit. Vasculogenic mimicry and laminin‐5γ2 double‐positive cohort displayed a significantly poorer disease‐free survival (DFS) and overall survival (OS). Vasculogenic mimicry, laminin‐5γ2 and their subsequent dual expression underlie a significant prognostic value for DFS [hazard ratio (HR) = 9.896, p =.028] and OS [HR = 21.401, p =.033] in OSCC patients. Together, our findings imply that VM along with laminin‐5γ2 is strongly linked to the malignant progression in OSCC and VM and laminin‐5γ2 coordination emerges as a critical prognostic biomarker for OSCC. [ABSTRACT FROM AUTHOR]

Abstract Vasculogenic mimicry, an endothelia-independent tumor microcirculation has been found in various cancers and is thought to be achieved by cancer stem like cells. Dacarbazine resistance is one of the most common features of melanoma and recent studies suggest that the mode of resistance is closely related to the formation of vasculogenic mimicry. In our work, we examined the anticancer effect of Lupeol, a novel phytochemical with Dacarbazine in vivo and in vitro. Results demonstrated adequate cytotoxicity followed by down regulation of CD 133 expression in Lupeol treated B16-F10 cell line. In solid tumor model the drug also inhibited vasculogenic mimicry along with angiogenesis by altering both the cancer stem cell as well as the endothelial progenitor cell population. Lupeol hindered the maturation of bone marrow derived endothelial progenitors and thus, retarded the formation of rudimentary tumor microvessels. Notably, Dacarbazine treatment demonstrated unresponsiveness to B16-F10 cells in both in vivo and in vitro model via upregulation of CD 133 expression and increased formation of vasculogenic mimicry tubes. Together, these data indicate that Lupeol alone can become a proficient agent in treating melanoma, inhibiting vasculogenic mimicry and might play a significant role in subduing Dacarbazine induced drug resistance. Highlights • Lupeol shows excellent cytotoxicity against B16-F10 melanoma cancer cells in vivo and in vitro. • Lupeol overcomes Dacarbazine resistance by inhibiting vasculogenic mimicry. • Lupeol also inhibits tumor angiogenesis by inhibiting the maturation and homing of Endothelial Progenitor Cells (EPC). [ABSTRACT FROM AUTHOR]

Background: Despite our efforts to develop effective treatment paradigms, the overall success rates of most of the anti-cancer therapies for aggressive cancers remain very low. There is an urgent need to develop novel therapeutics to meet the present challenges. In recent years, several plant derived chemicals showed promising return as antineoplastic agents. Objective: Here, we evaluate the anticancer effects of one well known phytochemical Lupeol on a panel of aggressive cancer types in preclinical platforms. Methods: Melanoma (B16- F10), breast cancer (MDA MB- 231), Cisplatin resistant laryngeal cancer cell line HEp-2c, oral cancer cell line UPCI: SCC131 and prostate cancer (PC-3) cell lines were treated with different doses of Lupeol for 24 and 48 hours. Cytotoxic effects of these agents were measured by different endpoint assays. Matrigel tube formation, Limiting Dilution (LD) and sphere formation assay along with alteration in CD133 expression were performed. The expression status of Ephrin A2 and its downstream molecules were examined in aggressive mouse melanoma solid tumor in vivo. We also established the key findings in a patient derived ex vivo explant model (CANScript TM) for human breast and HNSCC tumors. Results: Viability (MTT) assay showed significant IC50 value for each cell lines treated with Lupeol followed by CFU assay. Concurrently, induction of apoptosis was found to be prominent in treatment arm. We also observed down regulation of CD133 expression and disrupted tube formation in all cell lines that together ascertain their ability to inhibit vasculogenic mimicry (VM). The ability of Lupeol to interfere with cancer stem cell (CSC) like properties was also evident where sphere formation and LD profile demonstrated alteration in the CSC pools for majority. The machinery of these effects suggested its link with Ephrin A2 and its downstream elements driving VM in melanoma in vivo tumour model. Further validation of these data in patient derived CANScript ex vivo setting where efficacy of this drug was tested on the patient tumors clinically progressed on standard of care (SOC). Discussion and conclusion: Together, these data suggest the promising roles of phytochemical in treating diverse cancer indications at aggressive or late stages of the diseases where existing paradigms frequently show limited efficacy. [ABSTRACT FROM AUTHOR]