Over the last two decades, berries and berry bioactives, particularly anthocyanins and their aglycones anthocyanidins (Anthos) have demonstrated excellent anti-oxidant, anti-proliferative, apoptotic and anti-inflammatory properties. However, their physicochemical and pharmacokinetic limitations such as, low permeability, and poor oral bioavailability are considered as unfavorable properties for development as drugs. Therefore there is a need to develop systems for efficient systemic delivery and robust bioavailability. In this study we prepared nano-formulation of bilberry-derived Anthos using exosomes harvested from raw bovine milk. Exosomal formulation of Anthos enhanced antiproliferative and anti-inflammatory effects compared with the free Anthos against various cancer cells in vitro. Our data also showed significantly enhanced therapeutic response of exosomal-Anthos formulation compared with the free Anthos against lung cancer tumor xenograft in nude mice. The Anthos showed no signs of gross or systemic toxicity in wild-type mice. Thus, exosomes provide an effective alternative for oral delivery of Anthos that is efficacious, cost-effective, and safe, and this regimen can be developed as a non-toxic, widely applicable therapeutic agent.

Lung cancer is the leading cause of cancer-related deaths worldwide and in the United States. Despite recent advancements in treatment approaches, metastasis remains a major therapeutic challenge in lung cancer and explains the extremely poor prognosis. Epithelial to mesenchymal transition (EMT), a complex process of cellular reprogramming has become an attractive drug target because it plays a crucial role in the metastasis of non-small cell lung cancer (NSCLC). In the present study, we examined the effects of withaferin A (WFA), a plant-derived steroidal lactone on EMT in human NSCLC cell lines. First, we demonstrated that WFA displayed time- and concentration-dependent cytotoxicity on A549 and H1299 NSCLC cells. Then, cells were exposed to ≤ 0.5 µM WFA for ≤ 4 h to minimize cytotoxicity and determined its effects on EMT, cell adhesion, motility, migration, and invasion. EMT induction was performed by culturing cells in serum-free media containing TGFβ1 (5 ng/mL) and TNFα (25 ng/mL) for 48 h. We observed that pretreatment of cells with WFA inhibited cell adhesion, migration, and invasion of A549 and H1299 cells. Using western blot, immunofluorescence, and qRT-PCR analysis, we demonstrated that WFA suppressed TGFβ1 and TNFα-induced EMT in both cell lines. Mechanistically, WFA suppressed the phosphorylation and nuclear translocation of Smad2/3 and NF-κB in A549 and H1299 cells. Together, our study provides additional evidence demonstrating the inhibitory effects of WFA on EMT induction in NSCLC cells and further demonstrates the therapeutic potential of WFA against the metastasis in NSCLC.

Platinum-taxane combination chemotherapy still represents the standard of care for advanced non-small cell lung cancer (NSCLC) with no targetable driver mutations. However, the efficacy of these drugs has plateaued at 10-14 months primarily due to dose-limiting toxicity, chemoresistance, and metastasis. Here, we explored the effects of withaferin A (WFA) alone and in combination with paclitaxel (PAC) on the growth, proliferation, migration, and invasion of human NSCLC cells. We show that the sensitivity of H1299 and A549 cells to concomitant treatment with PAC and WFA was greater than that of either PAC or WFA alone. Using the combination index and dosereduction index, we demonstrated that various combinations (1:40, 1:20, 1:10) of PAC to WFA, respectively, were highly synergistic. In addition, PAC+WFA co-treatment synergistically inhibited colony formation, migration, invasion and increased the induction of apoptosis in H1299 and A549 cells. Interestingly, the synergism of PAC and WFA was not schedule-dependent but was enhanced when cells were pretreated with WFA indicating a chemo-sensitizing effect. Importantly, WFA was active against both PAC-sensitive (TS-A549) and PAC-resistant (TR-A549) cells both in vitro and in vivo. Mechanistically, WFA inhibits the proliferation of NSCLC cells via thiol oxidation. The effects of WFA were inhibited in the presence of N-acetyl cysteine and other thiol donors. Taken together, our results demonstrate the efficacy of WFA alone or alongside PAC on NSCLC cells and provide a strong rationale for further detailed testing in clinically relevant models for the development of PAC+WFA combination as an alternative therapeutic strategy for advanced NSCLC.

Background: Metastasis remains the primary cause in lung cancer. Epithelial-to-mesenchyme transition (EMT) has been identified as the hallmark for metastasis in NSCLC, and is being explored for drug targeting. Aberrant signaling mechanisms for cytokines such as TGF-β1 and TNFα are known to induce EMT. In this study we demonstrate the effect and mechanisms of WFA inhibition of EMT in NSCLC cell lines. Objective: The present study was done to determine the effect of WFA on TGF-β1 and TNFα induced EMT, migration and invasion in human NSCLC cells. Furthermore, to determine underlying the mechanisms of WFA inhibition of EMT. Methods: Two human NSCLC cell lines (H1299 and A549) were cultured in DMEM media. To induce the EMT, cells in serum free media were challenged with TGF-β1 (5 ng/mL) or TNFα (5 ng/mL) alone or in combination. To determine the effects of WFA, cells were either pre- or co-treated with different concentrations of WFA for various time points. Western blot analysis, electrophoretic mobility shift assay (EMSA), RT-PCR and immunofluorescence staining were used to determine effects of WFA on Smad2/3 and NFkB activation. Cell adhesion, migration and invasion assays were used to assess in vitro anti-metastatic activity. Results: Our findings indicate that the cytokines TGF-β1 and TNFα cooperate to promote the acquisition of EMT phenotype, migration and invasion of NSCLC cells in vitro. There was a marked increase in the expression of EMT transcription factors Snail, Slug, ZEB and EMT markers such as vimentin and N-cadherin. Cells treated with TGF-β1/TNFα had significantly increased migratory and invasive capacities. WFA treatment significantly inhibited the TGF-β1 induced Smad2/3 activation and TNFα-induced translocation of NFkB into the nucleus. We observed a dose and time dependent inhibition of the phosphorylation of Smad2/3 and NFkB in both cell lines. The cell adhesion, migratory and invasive capacities of both H1299 and A549 cells were greatly diminished by WFA in the presence or absence of cytokines. Conclusion: Based on the current finding, it can be concluded that WFA inhibits EMT via negative regulation of Smad2/3 and NFkB signaling in NSCLC cells. Citation Format: Al Hassan Kyakulaga, Farrukh Aqil, Radha Munagala, Ramesh Gupta. Withaferin A inhibits epithelial-to-mesenchymal transition in non-small lung cell cancer cells via regulation of SMAD and NFkB signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2040.

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