BackgroundProgrammed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Therefore, small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed.MethodsChanges of protein expression and phosphorylation levels were determined by immunoblotting. The level of Membrane PD-L1 was examined by flow cytometer. Cytotoxicity of T cells and NK cells toward tumor cells were detected using LDH and cell index assays. Lysosome function was investigated by NAG assay. Changes in lysosomal-related genes were measured by RT-PCR. In vivo anti-NSCLC cancer effects were assessed using C57BL/6 mice bearing Lewis tumor xenografts.FindingsWe identified SA-49 as a new regulator of PD-L1 expression from a series of novel aloperine derivatives. SA-49 decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells toward tumor cells. Importantly, lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3β activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice.InterpretationOur data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function.

The mitogen-activated protein kinase JNK1 suppresses interleukin-3 withdrawal-induced cell death through phosphorylation of the BH3-only pro-apoptotic Bcl-2 family protein Bad at Thr-201. It is unknown whether JNK1 regulates glycolysis, an important metabolic process that is involved in cell survival, and if so, whether the regulation depends on Thr-201 phosphorylation of Bad. Here we report that phosphorylation of Bad by JNK1 is required for glycolysis through activation of phosphofructokinase-1 (PFK-1), one of the key enzymes that catalyze glycolysis. Genetic disruption of Jnk1 alleles or silencing of Jnk1 by small interfering RNA abrogates glycolysis induced by growth/survival factors such as serum or interleukin-3. Proteomic analysis identifies PFK-1 as a novel Bad-associated protein. Although the interaction between PFK-1 and Bad is independent of JNK1, Thr-201 phosphorylation of Bad by JNK1 is required for PFK-1 activation. Thus, our results provide a novel molecular mechanism by which JNK1 promotes glycolysis for cell survival.The c-Jun N-terminal protein kinase (JNK; 2 also known as stress-activated protein kinase, SAPK) (1), a subfamily of the mitogen-activated protein kinase (MAPK) superfamily, has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform JNK3 with different splicing variant (2-4). Between JNK1 and JNK2, JNK1 is the main c-Jun kinase (5-7). JNK is activated by a variety of extracellular stimuli, including growth factors, cytokines, oncogenes, and environmental stresses through a MAP kinase module, i.e. MAP3K 3 MAP2K 3 MAPK (4). Two MAP2Ks (JNKK1/ MKK4/SEK1 and JNKK2/MKK7) for JNK have been identified (8 -11). Several MAP3Ks, such as MEKK1 (12), MEKK2 (13), apoptosis signal-regulating kinase (ASK1) (14), mixed lineage kinase (MLK) (15), TGF␤-activated kinase (TAK1) (16), tumor repression locus-2 (Tpl-2) (17), dual leucine zipper-bearing kinase (DLK) (18), and thousand and one amino acid kinase (Tao) (19), have been reported to act as MAP3Ks for JNK. Activation of JNK is also regulated by scaffold proteins such as JIP, ␤-arrestin, JSAP1 (20 -22), and protein phosphatases such as mitogen-activated protein phosphatases (23). In addition, the transcription factor NF-B can negatively or positively regulate JNK activation, depending on the nature of the stimulus (7, 24 -29). JNK plays a critical role in many cellular activities, from growth control to programmed cell death, and deregulation of JNK activity has been implicated in many human diseases and certain types of cancer (28, 30 -33).Bad is a BH3-only pro-apoptotic Bcl-2 family protein, having two splicing forms Bad s and Bad L (34). Bad plays a critical role in the cross-talk between the growth/survival factor signaling pathway and the intrinsic death machinery (35). The pro-apoptotic activity of Bad is regulated by extracellular stimuli via post-translational modifications including phosphorylation (35)(36)(37)(38). In response to the stimulation of growth/survival factors such as interleukin-3 (IL-3) and insul...

Targeting the autophagic pathway is currently regarded as an attractive strategy for cancer drug discovery. Our previous work showed that IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells, yet the effect of IMB-6G on autophagy and pancreatic cancer cell death remains unknown. Here, we show that IMB-6G inhibits the growth of MiaPaCa-2 and HupT-3 pancreatic cancer cells and induces caspase-mediated apoptosis, which is correlated with an accumulation of autophagic vacuoles. IMB-6G promotes autophagosome accumulation from the early stage of treatment but blocks autophagic flux in the degradation stage, mainly through attenuation of lysosomal cathepsin activity in pancreatic cancer cells. Moreover, IMB-6G triggers lysosomal membrane permeabilization (LMP), followed by cathepsin B/CTSB and cathepsin D/CTSD release from lysosomes into the cytoplasm. Inhibition of autophagosome formation with siRNA against autophagy protein 5 (Atg5) attenuates IMB-6G-induced LMP and apoptosis. Furthermore, cathepsin inhibitors relieve IMB-6G-induced apoptosis as well. Altogether, our findings demonstrate that IMB-6G is a novel autophagy inhibitor, which induces autophagy-dependent apoptosis through autophagosomal-cathepsin axis in pancreatic cancer cells and indicate the potential value of IMB-6G as a novel antitumor drug candidate.

Twenty-three new berberine (BBR) analogues defined on substituents of ring D were synthesized and evaluated for their activity for suppression of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation. Structure–activity relationship (SAR) analysis indicated that suitable tertiary/quaternary carbon substitutions at the 9-position or rigid fragment at position 10 might be beneficial for enhancing their anti-inflammatory potency. Among them, compounds 2d, 2e, 2i and 2j exhibited satisfactory inhibitory potency against NF-κB activation, with an inhibitory rate of around 90% (5 μM), much better than BBR. A preliminary mechanism study revealed that all of them could inhibit TNF-α-induced NF-κB activation via impairing IκB kinase (IKK) phosphorylation as well as cytokines interleukin (IL)-6 and IL-8 induced by TNF-α. Therefore, the results provided powerful information on further structural modifications and development of BBR derivatives into a new class of anti-inflammatory candidates for the treatment of inflammatory diseases.

No abstract

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.

No abstract

Sophoridinic acid derivatives have received considerable attentions for their potencies in cancer therapy. IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells. In the present study, we explored the antitumor abilities of IMB-6G in human hepatocellular carcinoma (HCC) cells and investigated the underlying mechanisms. We found that IMB-6G inhibited cell growth and induced mitochondrial-dependent apoptosis in HepG2 and SMMC7721 cells. Analyses of the molecular mechanism of IMB-6G-induced apoptosis indicated IMB-6G induced endoplasmic reticulum (ER) stress activation. Incubation of HCC cells with IMB-6G induced increase in Bip and CHOP levels, which precede induction of apoptosis. Further study showed IMB-6G activated IRE1α and PERK pathways but did not stimulated ATF6 pathway in HCC cells. Moreover, silencing of IRE1α dramatically abrogated IMB-6G-induced pro-apoptotic ASK1-JNK signaling. Importantly, interruption of CHOP rendered HCC cells sensitive to IMB-6G-induced apoptosis via inactivation of Bim, PUMA and Bax. Thus, the IRE1α-ASK1 and PERK-CHOP pathways may be a novel molecular mechanism of IMB-6G-induced apoptosis. Collectively, our study demonstrates that IMB-6G induces ER stress-mediated apoptosis by activating IRE1α and PERK pathways. Our findings provide a rationale for the potential application of IMB-6G in HCC therapy.