Synergistic killing effect of paclitaxel and honokiol in non-small cell lung cancer cells through paraptosis induction.

PURPOSE: Paclitaxel is an anticancer drug for the treatment of non-small cell lung cancer (NSCLC). However, drug-resistance remains a major problem. Honokiol is a natural component which has been found to exhibit anti-tumor activity. Paclitaxel and honokiol have been reported to be able to induce paraptosis. The aim of this study was to investigate whether honokiol can reverse paclitaxel resistance by inducing paraptosis in NSCLC cells. METHODS: NSCLC cell lines H1650 (paclitaxel-sensitive), H1299 and H1650/PTX (intrinsic and acquired paclitaxel-resistant, respectively) were used to assess the cytotoxic effects of paclitaxel and honokiol. Light and transmission electron microscopy were performed to detect cytoplasmic vacuolation. In vitro cell viability and clonogenic survival assays, as well as in vivo xenograft assays were conducted to test synergistic killing effects of paclitaxel and honokiol on NSCLC cells. Western blotting, flow cytometry and immunofluorescence were performed to evaluate paraptosis-regulating mechanisms. RESULTS: We found that combination treatment with paclitaxel and honokiol synergistically killed H1650, H1299 and H1650/PTX cells by inducing paraptosis, which is characterized by cytoplasmic vacuolation. Moreover, paclitaxel/honokiol treatment resulted in a significant growth delay in H1299 xenograft tumors that showed extensive cytoplasmic vacuolation. Mechanistically, proteasomal inhibition-mediated endoplasmic reticulum (ER) stress and unfolded protein responses leading to ER dilation, and the disruption of intracellular Ca2+ homeostasis and mitochondrial Ca2+ overload resulting in mitochondrial disfunction, were found to be involved in paclitaxel/honokiol-induced paraptosis. Cellular protein light chain 3 (LC3) may play an important role in paclitaxel/honokiol induced cytoplasmic vacuolation and NSCLC cell death. CONCLUSIONS: Combination of honokiol and paclitaxel may represent a novel strategy for the treatment of paclitaxel-resistant NSCLC.

Acquired tamoxifen resistance is surmounted by GW8510 through ribonucleotide reductase M2 downregulation-mediated autophagy induction.

Tamoxifen resistance is a major roadblock in the treatment of patients with breast cancer. Ribonucleotide reductase M2 (RRM2) was found to be involved in acquired resistance of breast cancer cells (BCCs) to tamoxifen. Here, we used GW8510, which has been identified as a potential RRM2 inhibitor, to evaluate the effect of RRM2 inhibition on reversing resistance of BCCs to tamoxifen and investigate its mechanisms. We showed that RRM2 overexpression played a key role in the development of acquired tamoxifen resistance in BCCs through downregulation of autophagy level. Combination treatment with tamoxifen and GW8510 significantly inhibited survival of the tamoxifen-resistant BCCs through induction of autophagic cell death compared to either of the two drugs. Furthermore, combination of tamoxifen and GW8510 resulted in marked growth inhibition of tamoxifen-resistant BBC xenograft tumor in vivo compared to tamoxifen or GW8510 alone. In conclusion, tamoxifen in combination with GW8510 can overcome acquired tamoxifen resistance in BCCs and may be a rational therapeutic approach against breast cancer with high RRM2 expression.

Curcumin overcome primary gefitinib resistance in non-small-cell lung cancer cells through inducing autophagy-related cell death.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are being wildly used as target therapy in non-small-cell lung cancer (NSCLC). However, NSCLC patients with wild-type EGFR and KRAS mutation are primary resistant to EGFR-TKIs such as gefitinib. Curcumin has been known as a potential therapeutic agent for several major human cancers. In this study, we investigated the effect of curcumin on the reversal of gefitinib resistance in NSCLC cells as well as their molecular bases. METHODS: H157 (wild-type EGFR and KARS mutation) and H1299 (wild-type EGFR and HRAS mutation) cells were treated with gefitinib or curcumin alone, or the two combination, and then cell viability, EGFR activity, expressions of Sp1 and Sp1-dependent proteins and receptor tyrosine kinases, markers of autophagy and apoptosis were examined by using CCK-8, colony formation, immunoblot, quantitative PCR, immunofluoscence, and flow cytometry assays. Also xenograft experiments were conduced to test the synergism of curcumin to gefitinib. RESULTS: Our results showed that curcumin significantly enhanced inhibitory effect of gefitinib on primary gefitinib-resistant NSCLC cell lines H157 and H1299. Combination treatment with curcumin and gefitinib markedly downregulated EGFR activity through suppressing Sp1 and blocking interaction of Sp1 and HADC1, and markedly suppressed receptor tyrosine kinases as well as ERK/MEK and AKT/S6K pathways in the resistant NSCLC cells. Meanwhile, combination treatment of curcumin and gefitinib caused dramatic autophagy induction, autophagic cell death and autophagy-mediated apoptosis, compared to curcumin or gefitinib treatment alone, as evidenced by the findings that curcumin and gefitinib combination treatment-produced synergistic growth inhibition and apoptosis activation can be reversed by pharmacological autophagy inhibitors (Baf A1 or 3-MA) or knockdown of Beclin-1 or ATG7, also can be partially returned by pan-caspase inhibitor (Z-VAD-FMK) in H157 and H1299 cells. Xenograft experiments in vivo yielded similar results. CONCLUSIONS: These data indicate that the synergism of curcumin on gefitinib was autophagy dependent. Curcumin can be used as a sensitizer to enhance the efficacy of EGFR-TKIs and overcome the EGFR-TKI resistance in NSCLC patients with wild-type EGFR and/or KRAS mutation.

Gemcitabine resistance mediated by ribonucleotide reductase M2 in lung squamous cell carcinoma is reversed by GW8510 through autophagy induction.

Although chemotherapeutic regimen containing gemcitabine is the first-line therapy for advanced lung squamous cell carcinoma (LSCC), gemcitabine resistance remains an important clinical problem. Some studies suggest that overexpressions of ribonucleotide reductase (RNR) subunit M2 (RRM2) may be involved in gemcitabine resistance. We used a novel RRM2 inhibitor, GW8510, as a gemcitabine sensitization agent to investigate the therapeutic utility in reversing gemcitabine resistance in LSCC. Results showed that the expressions of RRM2 were increased in gemcitabine intrinsic resistant LSCC cells upon gemcitabine treatment. GW8510 not only suppressed LSCC cell survival, but also sensitized gemcitabine-resistant cells to gemcitabine through autophagy induction mediated by RRM2 down-regulation along with decrease in dNTP levels. The combination of GW8510 and gemcitabine produced a synergistic effect on killing LSCC cells. The synergism of the two agents was impeded by addition of autophagy inhibitors chloroquine (CQ) or bafilomycin A1 (Baf A1), or knockdown of the autophagy gene, Bcl-2-interacting protein 1 (BECN1). Moreover, GW8510-caused LSCC cell sensitization to gemcitabine through autophagy induction was parallel with impairment of DNA double-strand break (DSB) repair and marked increase in cell apoptosis, revealing a cross-talk between autophagy and DNA damage repair, and an interplay between autophagy and apoptosis. Finally, gemcitabine sensitization mediated by autophagy induction through GW8510-caused RRM2 down-regulation was demonstrated in vivo in gemcitabine-resistant LSCC tumor xenograft, further indicating that the sensitization is dependent on autophagy activation. In conclusion, GW8510 can reverse gemcitabine resistance in LSCC cells through RRM2 downregulation-mediated autophagy induction, and GW850 may be a promising therapeutic agent against LSCC as it combined with gemcitabine.