Chunping Gu

Induction of autophagy counteracts the anticancer effect of cisplatin in human esophageal cancer cells with acquired drug resistance

Cisplatin-based chemotherapy frequently resulted in acquired resistance. The underpinning mechanism of such resistance remains obscure especially in relation to autophagic response. This study thus investigated the role of autophagy in the anticancer activity of cisplatin in human esophageal cancer cells with acquired cisplatin resistance. In response to cisplatin treatment, EC109 cells exhibited substantial apoptosis and senescence whereas cisplatin-resistant EC109/CDDP cells exhibited resistance. In this respect, cisplatin increased ERK phosphorylation whose inhibition by MEK inhibitor significantly attenuated the cytotoxic and cytostatic effect of cisplatin. Notably, cisplatin preferentially induces autophagy in EC109/CDDP cells but not in EC109 cells. Moreover, the induction of autophagy was accompanied by the suppression of mTORC1 activity. Abolition of autophagy by pharmacological inhibitors or knockdown of ATG5/7 re-sensitized EC109/CDDP cells. Co-administration of an autophagy inhibitor chloroquine and cisplatin significantly suppressed tumor growth whereas cisplatin monotherapy failed to elicit anticancer activity in nude mice xenografted with EC109/CDDP cells. To conclude, our data implicate autophagic response as a key mechanism of acquired resistance to cisplatin, suggesting that autophagy is a novel target to improve therapy efficiency of cisplatin toward human esophageal cancers with acquired resistance.

Sinomenine Suppresses Osteoclast Formation and Mycobacterium tuberculosis H37Ra-Induced Bone Loss by Modulating RANKL Signaling Pathways

Receptor activator of NF-κB ligand (RANKL) is essential for osteoclastogenesis. Targeting RANKL signaling pathways has been an encouraging strategy for treating lytic bone diseases such as osteoporosis and rheumatoid arthritis (RA). Sinomenine (SIN), derived from Chinese medicinal plant Sinomenioum acutum , is an active compound to treat RA, but its effect on osteoclasts has been hitherto unknown. In the present study, SIN was found to ameliorate M. tuberculosis H37Ra (Mt)-induced bone loss in rats with a decreased serum level of TRACP5b and RANKL, and an increased level of osteoprotegerin (OPG). In vitro study also showed that SIN could inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, MMP-9, TRACP were inhibited by SIN in a dose dependent manner. Signal transduction studies showed that SIN could obviously reduce the expression of RANK adaptor molecule TRAF6 and down-regulate RANKL-induced NF-κB activation. It decreased the RANKL-induced p38, JNK posphorylation but not ERK1/2 posphorylation. SIN could also reduce RANKL-mediated calcium influx which is associated with TRAF6/c-Src complex. Finally, SIN suppressed RANKL induced AP-1 and NFAT transcription, as well as the gene expression of NFATc1 and AP-1 components (Fra-1, Fra-2, c-Fos). The protein expression of c-Fos and TRAF6 were also inhibited by SIN after RANKL stimulation. Taken together, SIN could attenuate osteoclast formation and Mt-induced bone loss by mediating RANKL signaling pathways.

Obatoclax Induces G1/G0‐Phase Arrest via p38/p21waf1/Cip1 Signaling Pathway in Human Esophageal Cancer Cells

Pan‐Bcl‐2 family inhibitor obatoclax has been demonstrated to be effective against various cancers, of which the mechanism of action is not fully understood. In this study, we demonstrate that obatoclax suppressed esophageal cancer cell viability with concomitant G1/G0‐phase cell cycle arrest. At the tested concentrations (1/2 IC50 and IC50), obatoclax neither induced PARP cleavage nor increased the Annexin V‐positive population, suggesting G1/G0‐phase arrest rather than apoptosis accounts for most of the reduction of cell viability produced by obatoclax. Double knockdown of Bak and Bax by small interference RNA failed to block obatoclax‐induced G1/G0‐phase arrest, implying its role in cell cycle progression is Bak/Bax‐independent. The cell cycle arresting effect of obatoclax was associated with up‐regulation of p21waf1/Cip1. Knockdown of p21waf1/Cip1 significantly attenuated obatoclax‐induced G1/G0‐phase arrest. Although obatoclax stimulated phosphorylation of Erk, p38, and JNK, pharmacological inhibition of p38 but not Erk or JNK blocked obatoclax‐induced G1/G0‐phase arrest. Moreover, knockdown of p38 abolished the cell cycle arresting effect of obatoclax. In consistent with this finding, inhibition of p38 blocked obatoclax‐induced p21waf1/Cip1 expression while inhibition of Erk or JNK failed to exert similar effect. To conclude, these findings suggest that obatoclax induced cell cycle arrest via p38/p21waf1/Cip1 signaling pathway. This study may shed a new light on the anti‐cancer activity of obatoclax in relation to cell cycle arrest. J. Cell. Biochem. 115: 1624–1635, 2014.

Celecoxib antagonizes the cytotoxic effect of cisplatin in human gastric cancer cells by decreasing intracellular cisplatin accumulation

Cisplatin is a chemotherapeutic drug widely used for the treatment of gastric cancer. The benefit of including COX-2-selective inhibitors in cisplatin-based regimens on anti-cancer effect remains uncertain. In the present study, celecoxib and SC-236 antagonized cisplatin-induced cytotoxicity and apoptosis, whereas indomethancin and nimesulide exerted no effect, implying a COX-2-independent mechanism. Celecoxib decreased whole-cell cisplatin accumulation and DNA platination, resulting from reduced influx. In addition, combined treatment did not elicit greater antitumor activity than cisplatin or celecoxib monotherapy in vivo in a gastric xenograft model. Therefore, treatment strategies with celecoxib in combination with cisplatin should act cautiously.

Obatoclax impairs lysosomal function to block autophagy in cisplatin-sensitive and -resistant esophageal cancer cells

Obatoclax, a pan-inhibitor of anti-apoptotic Bcl-2 proteins, exhibits cytotoxic effect on cancer cells through both apoptosis-dependent and -independent pathways. Here we show that obatoclax caused cytotoxicity in both cisplatin-sensitive and -resistant esophageal cancer cells. Although obatoclax showed differential apoptogenic effects in these cells, it consistently blocked autophagic flux, which was evidenced by concomitant accumulation of LC3-II and p62. Obatoclax was trapped in lysosomes and induced lysosome clustering. Obatoclax also substantially reduced the expression of lysosomal cathepsins B, D and L. Moreover, cathepsin knockdown was sufficient to induce cytotoxicity, connecting lysosomal function to cell viability. Consistent with the known function of autophagy, obatoclax caused the accumulation of polyubiquitinated proteins and showed synergy with proteasome inhibition. Taken together, our studies unveiled impaired lysosomal function as a novel mechanism whereby obatoclax mediates its cytotoxic effect in esophageal cancer cells.

Celecoxib antagonizes the cytotoxicity of oxaliplatin in human esophageal cancer cells by impairing the drug influx.

It has been demonstrated that COX-2-selective inhibitor celecoxib shows synergy with oxaliplatin for suppressing tumor growth. However, the benefit of adding celecoxib to oxaliplatin-based regimen in human esophageal cancer is largely unknown. In the present study, we demonstrated that celecoxib antagonized oxaliplatin-induced cytotoxicity and apoptosis independent of COX-2 inhibition in human esophageal cancer cells. Celecoxib decreased cellular oxaliplatin accumulation and Pt-DNA adduction formation due to reduced drug influx. Celecoxib alone or combined with oxaliplatin substantially reduced the expression of organic cation transporter 2 (OCT2). To this end, OCT2 knockdown was sufficient to reduce oxaliplatin uptake, connecting OCT2 expression to oxaliplatin accumulation. Moreover, oxaliplatin combined with celecoxib also showed no beneficial effect when compared with monotherapy in esophageal cancer cell-xenografted nude mice. To conclude, our data provide evidence that the addition of celecoxib to oxaliplatin-containing regimens for patients with OCT2-expressing cancers should be cautious.

A novel synthetic dibenzocyclooctadiene lignan analog XLYF-104-6 attenuates lipopolysaccharide-induced inflammatory response in RAW264.7 macrophage cells and protects BALB/c mice from sepsis

The wide range of inflammation mechanisms under control by NF-κB makes this pathway as an attractive target for new anti-inflammatory drugs. Herein, we showed that a new dibenzocyclooctadiene lignan analog XLYF-104-6, with a chemical name of 1,2,3,10,11-pentamethoxydibenzocycloocta-6,7-[c] pyrrole-1,3-dione, inhibited lipopolysaccharide (LPS)-induced NF-κB activation in RAW264.7 cells through preventing IκBα degradation and p65 nuclear translocation. The inhibitory activity of this compound on NF-κB activation contributes to the reduction of LPS-induced TNF-α and IL-6 productions. Notably, XLYF-104-6 suppressed LPS-induced iNOS expression and NO production in a NF-κB independent manner, since IKK inhibitor BAY 11-7082 has failed to exert similar inhibitory effect on iNOS expression and NO production. In addition, XLFY-104-6 also exerted anti-inflammatory action in endotoxemic mice by decreasing plasma LPS-induced TNF-α and IL-1β levels as well as increasing plasma LPS-induced IL-10 concentrations. These findings suggest XLYF-104-6 could act as a leading compound for developing a potential anti-inflammatory drug.

Synthesis, selective cytotoxicities and probable mechanism of action of 7-methoxy-3-arylflavone-8-acetic acids

Thirteen new analogues of flavone-8-acetic acid, that is, compounds 10a-m bearing a methoxy group at the 7-position and diverse subsitiuents on the benzene ring at the 2- and 3-positions of flavone nucleus, were synthesized and evaluated for their direct antiproliferative effects on two human tumor cell lines and for their indirect antiproliferative activities in the transwell co-culture system. The results indicated that most of compounds 10a-m showed moderate direct cytotoxicities. Among them, compound 10i exhibited higher direct cytotoxicity and selectivity for both cell lines over BJ human foreskin fibroblast cells than 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Interestingly, compared with DMXAA, compound 10e showed comparable indirect cytotoxicity and higher selectivity. In addition, compound 10e was found to be able to induce tumor necrosis factor α (TNF-α) production in human peripheral blood mononuclear cells.

Discovery of novel antitumor dibenzocyclooctatetraene derivatives and related biphenyls as potent inhibitors of NF-κB signaling pathway.

Several dibenzocyclooctatetraene derivatives (5-7) and related biphenyls (8-11) were designed, synthesized, and evaluated for inhibition of cancer cell growth and the NF-κB signaling pathway. Compound 5a, a dibenzocyclooctatetraene succinimide, was discovered as a potent inhibitor of the NF-κB signaling pathway with significant antitumor activity against several human tumor cell lines (GI₅₀ 1.38-1.45 μM) and was more potent than paclitaxel against the drug-resistant KBvin cell line. Compound 5a also inhibited LPS-induced NF-κB activation in RAW264.7 cells with an IC₅₀ value of 0.52 μM, prevented IκB-α degradation and p65 nuclear translocation, and suppressed LPS-induced NO production in a dose-dependent manner. The antitumor data in cellular assays indicated that relative positions and types of substituents on the dibenzocyclooctatetraene or acyclic biphenyl as well as torsional angles between the two phenyls are of primary importance to antitumor activity.

ABT-263 induces G 1 /G 0 -phase arrest, apoptosis and autophagy in human esophageal cancer cells in vitro

Both the anti- and pro-apoptotic members of the Bcl-2 family are regulated by a conserved Bcl-2 homology (BH3) domain. ABT-263 (Navitoclax), a novel BH3 mimetic and orally bioavailable Bcl-2 family inhibitor with high affinity for Bcl-xL, Bcl-2 and Bcl-w has entered clinical trials for cancer treatment. But the anticancer mechanisms of ABT-263 have not been fully elucidated. In this study we investigated the effects of ABT-263 on human esophageal cancer cells in vitro and to explore its anticancer mechanisms. Treatment with ABT-263 dose-dependently suppressed the viability of 3 human esophageal cancer cells with IC50 values of 10.7±1.4, 7.1±1.5 and 8.2±1.6 μmol/L, in EC109, HKESC-2 and CaES-17 cells, respectively. ABT-263 (5-20 μmol/L) dose-dependently induced G1/G0-phase arrest in the 3 cancer cell lines and induced apoptosis evidenced by increased the Annexin V-positive cell population and elevated levels of cleaved caspase 3, cleaved caspase 9 and PARP. We further demonstrated that ABT-263 treatment markedly increased the expression of p21Waf1/Cip1 and decreased the expression of cyclin D1 and phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780) proteins that contributed to the G1/G0-phase arrest. Knockdown of p21Waf1/Cip1 attenuated ABT-263-induced G1/G0-phase arrest. Moreover, ABT-263 treatment enhanced pro-survival autophagy, shown as the increased LC3-II levels and decreased p62 levels, which counteracted its anticancer activity. Our results suggest that ABT-263 exerts cytostatic and cytotoxic effects on human esophageal cancer cells in vitro and enhances pro-survival autophagy, which counteracts its anticancer activity.