The data presented here show that treatment with OSI resulted in substantial induction of apoptosis and anti-proliferation activity in EGFR mutations compared to WT controls. In EGFR wild-type NSCLC cells, expression of cleaved-caspase 3 and TUNEL assays revealed no changes in apoptosis and anti-proliferative activity after treatment with OSI. However, following the addition of pantoprazole, the effects of OSI on NSCLC cells increased significantly, suggesting that inhibition of autophagy by pantoprazole represents a promising option for enhancing OSI efficacy in NSCLC.
In a previous study, it was reported that OSI induced autophagy in various cancer cell lines, including NSCLC H1975, HCC827, and A549 cells, as well as ovarian, colon and hepatocellular cells 13. These observations were broadly consistent with our results, with OSI inducing autophagy of NSCLC cells both in vivo and in vitro. OSI-induced autophagy in A549 cells and EGFR WT xenograft models showed that EGFR-TKIs may induce autophagy independent of EGFR mutations. Decreases in OSI-induced cell viability were largely reversed following addition of pantoprazole, implying that inhibition of autophagy increased cytotoxicity of OSI in vivo and in vitro, regardless of EGFR mutation. Autopahgy inhibition may increase cytotoxicity of OSI by downregulating the expression SOX2 and aldehyde dehydrogenase 1A1 (ALDH1A1) 22.
Autophagy is typically regarded as a protective mechanism that is capable of promoting cellular survival under unfavorable conditions such as deprivation of ATPs, particularly in stressed cells, including poorly nourished tumor cells 15. Cancer cells use autophagy as a survival mechanism to counter the stress caused by chemotherapy, making autophagy an appropriate target for improving the activity of anticancer drugs 20. Our data are consistent with other recent studies that have demonstrated that resistance to anticancer therapy is associated with up-regulation of autophagy 23, 24. One such study found that cisplatin and paclitaxel were able to induce autophagy in A549 lung cancer cells, while inhibition of autophagy promoted cisplatin and paclitaxel-induced apoptosis 25. It has also been reported that disulfiram/copper may be able to stimulate autophagy in NSCLC cells, which is alternatively enhanced and suppressed via inhibition using Atg5 siRNA or enhanced by the addition of 3-MA 26. Acute autophagy ablation in mice with pre-existing NSCLC blocked tumor growth, and promoted tumor cell death, suggesting that acute autophagic inhibition may have anticancer activity 27. Several anticancer agents have been shown to induce the accumulation of autophagosomes, which is an initial step in the autophagic pathway 28. These approaches led to poor clinical outcomes 29 with a high chance of acquiring development of resistance 30, highlighting the need for alternative therapeutic options.
Proton pump inhibitors have been reported to sensitize cancer cells and solid tumors to different chemotherapeutic agents. One study implied that pantoprazole inhibited autophagy by raising pH in lysosomes that fuse with autophagosomes, and/or inhibiting this fusion directly 19. These results are strongly supported by our results, which showed that pantoprazole increased the accumulation of p62 and inhibited the conversion of LC3-I to LC3-II. The steady-state levels of the p62 protein can reflect autophagic status, and p62 levels increase when autophagy is blocked 31. Therefore, our results indicate that pantoprazole can inhibit autophagy in NSCLC.
It has been reported that deletion of Atg5 inhibited autophagy and enhanced norcantharidin-induced apoptosis in hepatocellular carcinoma 32. Stable knockdown of Atg5 by siRNA transfection impaired cisplatininduced activation of autophagic responses, increased caspase-3 cleavage and inhibited cell viability in A549 cells 33. To prove the mechanism of autophagy, we used Atg5 siRNA transfection to knock down Atg5 in A549 and H1975 cells. The present study found increased ROS in Atg5 siRNA transfected cells in which autophagy was decreased. Knockdown of Atg5 increased pantoprazole-induced cytotoxicity of OSI, presumably due to the combined effects of abrogate autophagy.
ROS, a class of oxygen-containing free-radicals, have an important effect on cellular processes, including autophagy and apoptosis 34. Inhibition of autophagy by knockdown of Atg 5 expression significantly increased ROS in OSI-treated cells. OSI-induced generation of ROS was enhanced after combination treatment with pantoprazole. The data imply that pantoprazole inhibited OSI-induced autophagy following oxidative stress in NSCLC cells.
Our results indicate that OSI-induced autophagy may be EGFR independent. OSI induced an autophagic response in both EGFR WT and EGFR mutation. The PI3K/Akt/mTOR pathway plays an important role in cell growth, survival, differentiation, and metabolism 35. This pathway regulates autophagy and apoptosis using a negative feedback loop 36, 37. The present results indicate that OSI reduced expression of PI3K, p-AKT, and p-mTOR, which was reversed after treatment with pantoprazole. Pantoprazole inhibited OSI-induced autophagy through modulation of the PI3K/AKT/mTOR pathway in NSCLC cells.
The absorption of EGFR-TKIs is affected by gastric pH value. The intestinal absorption of erlotinib is impaired under PPI administration 38. When erlotinib and a PPI are given concomitantly, the plasma concentration of erlotinib steeply decreases, which suggests that lower bioavailability due to PPI use may deprive patients from optimal therapy 39, 40. Therefore, more findings need to be used to optimize the management of PPI and EGFR-TKIs.
For some TKIs, the question remains whether taking PPI along with TKI would increase or decrease the aborption of TKI. However, this is widely studied. So far, two studies reported coadministration with PPI did not significantly affect OSI exposure 41, 42. Another study has shown maximum concentration of OSI decreased after co-administration with rabeprazole 43. In our study, we found that the tumor suppression effect of OSI in combination with pantoprazole became stronger than that of OSI alone. However, drug-drug interactions in vivo were indeed not confirmed, which is a shortcoming of our study.
In conclusion, enhaced autophagy may be a cause of OSI resistance. This study demonstrated that pantoprazole invokes strong anti-cancer activity of OSI against NSCLC by enhancing OSI-induced apoptosis as well as autophagy through regulating the PI3K/AKT/mTOR pathway and ROS generation. These findings provide a strong rationale for upcoming combination therapies including PPI and EGFR-TKIs for the treatment of NSCLC. The combination OSI and pantoprazole has been shown to be feasible with good toxicity profile. Further clinical trals are needed to assess the efficacy of pantoprazole combined with OSI in patients with NSCLC.