br Inhibition of autophagy sensitizes
3.3. Inhibition of autophagy sensitizes parental and resistant Pam3CSK4 to icotinib
To determine whether autophagy inhibition sensitizes cells to ico-tinib, we evaluated the combined eﬀects of chloroquine and icotinib on cell proliferation using parental and resistant cells. As shown in Fig. 3 A and B, CQ significantly augmented the icotinib-induced growth in-hibition. For further confirmation, we specifically inhibited autophagy with siRNAs targeting essential ATG 6 (Beclin-1) (Fig. 3 C). Consistent with experimental results using pharmacological inhibitors, autophagy inhibition by Atg 6 knockdown significantly decreased viability in both parental and resistant cells under icotinib treatment (Fig. 3 D and E). These results suggest that the combination of autophagy inhibition and icotinib is more eﬀective compared with either treatment alone in both icotinib-sensitive and icotinib-resistant cells.
3.4. Rg3 inhibits autophagic flux and sensitizes parental and resistant cells to icotinib
Several studies have suggested that Rg3 can inhibit autophagic flux. We examined the eﬀect of Rg3 on autophagy by measuring LC3 II protein levels in Rg3-treated cells. Rg3 treatment of PC-9 and HCC827 cells increased the level of LC3 II protein in a dose- and time-dependent manner (Fig. 4 A and B). We also observed increased numbers of LC3 II puncta in PC-9 and HCC827 cells treated with Rg3 for 24 h compared with their controls (Fig. 4 C and D). At the same time, Rg3 treatment led to an increase in p62 (Fig. 4 E). An increase in LC3 II may represent either increased generation of autophagosomes and/or inhibition of autophagosomal degradation. A decrease in p62 can be a marker for an increase in autophagic flux. However, Rg3 treatment led to an increase in p62. Stabilization of p62 levels suggests that Rg3 inhibits its de-gradation. Additionally, cells treated with CQ and Rg3 did not have substantially enhanced LC3 II and p62 protein levels (Fig. 4 E). This observation suggests that the increased LC3 puncta formation accom-panied by increased p62 levels in Rg3-treated cells may be due to suppression of the late maturation or degradation stage of autophagy.
To determine whether the suppression of autophagic flux by Rg3 sensitizes cells to icotinib, we evaluated the combined eﬀects of Rg3 and icotinib on cell proliferation using parental and resistant cells. As shown in Fig. 5 A-D, Rg3 significantly augmented the icotinib-induced growth inhibition in both parental and resistant cells. These results indicate that Rg3 can sensitize NSCLC cells to icotinib and reverse the resistance to icotinib through inhibition of autophagy.
3.5. Rg3 reverses resistance to icotinib in an in vivo mouse model
To further determine the therapeutic eﬀect of Rg3 in combination with icotinib, we established PC-9R xenograft models in nude mice. As shown in Fig. 6 A and B, Rg3 alone and icotinib alone had no eﬀect on the growth of tumors. Consistent with the in vitro results, combination treatment with Rg3 and icotinib significantly reduced tumor growth, indicating that Rg3 reverses the resistance to icotinib in vivo. Im-munohistochemical (IHC) analysis showed that the combination treat-ment decreased the expression of proliferating cell nuclear antigen (PCNA) but increased the expression of LC3-II (Fig. 6 C). Moreover, no significant weight loss or liver and kidney injury was observed due to
Fig. 6. The combination of Rg3 plus icotinib reverses the resistance to icotinib in a mouse xenograft model. PC-9R tumors were established and treated with Rg3, icotinib or Rg3 plus icotinib for 14 days. Tumor volume (A) and weight (B) in each group. C, Immunohistochemical staining of LC3 and PCNA using paraﬃn-embedded sections.