Epidermal Growth Factor Receptor (EGFR)-targeted Photoimmunotherapy (PIT) for the Treatment of EGFR-expressing Bladder Cancer.

The use of light as a means of therapy for bladder cancer has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT), which targets EGFR-expressing bladder cancer. Anti-EGFR antibody panitumumab was labeled with the photoabsorber (PA), IRDye 700Dx (IR700), to create a panitumumab-IR700 antibody-PA conjugate that is activated by near-infrared radiation (NIR). Bladder cancer tissue microarray (TMA) and bladder cancer cell lines were analyzed for expression of EGFR. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most bladder cancers have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Panitumumab-IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Panitumumab alone, panitumumab-IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed toward cell death. NIR-PIT with panitumumab-IR700 reduced growth compared with only panitumumab-IR700-treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Panitumumab-IR700-induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of bladder cancer and ultimately in patients. Mol Cancer Ther; 16(10); 2201-14. ©2017 AACR.

ß-Elemene promotes cisplatin-induced cell death in human bladder cancer and other carcinomas.

Cisplatin-based combination treatment is the most effective systemic chemotherapy for bladder cancer; however, resistance to cisplatin remains a significant problem in the treatment of this disease. ß-Elemene is a new natural compound that blocks cell-cycle progression and has a broad spectrum of antitumor activity. This study was conducted to explore the potential of ß-elemene as a chemosensitizer for enhancing the therapeutic efficacy and potency of cisplatin in bladder cancer and other solid carcinomas. ß-Elemene not only markedly inhibited cell growth and proliferation but also substantially increased cisplatin cytotoxicity towards human bladder cancer 5637 and T-24 cells. Similarly, ß-elemene also enhanced cisplatin sensitivity and augmented cisplatin cytotoxicity in small-cell lung cancer and carcinomas of the brain, breast, cervix, ovary, and colorectal tract in vitro, with dose-modifying factors ranging from 5 to 124. ß-Elemene-enhanced cisplatin cytotoxicity was associated with increased apoptotic cell death, as determined by DNA fragmentation, and increased activities of caspase-3, -7, -8, -9, and -10 in bladder cancer cell lines. Collectively, these results suggest that ß-elemene augments the antitumor activity of cisplatin in human bladder cancer by enhancing the induction of cellular apoptosis via a caspase-dependent mechanism. Cisplatin combined with ß-elemene as a chemosensitizer warrants further pre-clinical therapeutic studies and may be useful for the treatment of cisplatin-resistant bladder cancer and other types of carcinomas.

ß-Elemene and taxanes synergistically induce cytotoxicity and inhibit proliferation in ovarian cancer and other tumor cells.

ß-Elemene, originally derived from plants, has been recently investigated as a new anticancer agent. The purpose of this study was to explore the efficacy and mechanisms of action of the combined use of ß-elemene plus a taxane as an antitumor therapeutic strategy for ovarian cancer and other carcinomas. The interaction of ß-elemene with paclitaxel or docetaxel produced additive to moderately synergistic effects against the platinum-resistant ovarian cancer cell line A2780/CP70 and its parental cell line A2780, and showed moderately synergistic activity against PC-3 prostate cancer cells. In addition, the co-administration of ß-elemene and a taxane at low-micromolar concentrations dramatically increased the rate of micronucleus formation and the percentage of mitotic arrest in both ovarian cancer cell lines, as compared with treatment with either agent alone. The highest synergy towards the ovarian cancer cells was observed with ß-elemene plus docetaxel. Consistent with these data, treatment of A2780/CP70 cells with ß-elemene plus a taxane strikingly reduced cell viability and increased cell apoptosis, as assessed by annexin V binding. Moreover, ß-elemene plus docetaxel induced elevated levels of caspase-9 and p53 proteins in A2780/CP70 cells, and the combination of ß-elemene plus a taxane caused marked cell-cycle arrest at the G2/M phase in these cells. One possible mechanism to account for the enhanced cytotoxic efficacy of this combination treatment is a ß-elemene-induced increase in taxane influx into cancer cells. These observations indicate that combination therapy with ß-elemene and taxanes has synergistic antitumor activity against ovarian and prostate carcinomas in vitro. This promising new therapeutic combination warrants further pre-clinical exploration for the treatment of chemoresistant ovarian cancer and other types of tumors.

Sensitization of lung cancer cells to cisplatin by ß-elemene is mediated through blockade of cell cycle progression: antitumor efficacies of ß-elemene and its synthetic analogs.

The development of effective agents for overcoming platinum chemoresistance in lung carcinoma continues to have high priority. We have demonstrated recently that ß-elemene, a novel antitumor compound, enhances cisplatin activity by triggering lung cancer cell death via apoptosis. Here, we investigated whether ß-elemene acts synergistically with cisplatin to inhibit non-small cell lung cancer (NSCLC) cell proliferation by blocking cell cycle progression. ß-Elemene substantially increased the suppressive effect of cisplatin on cell growth and proliferation in the NSCLC cell lines H460 and A549. Furthermore, ß-elemene augmented cisplatin in the cell cycle arrest of NSCLC cells at G(2)/M. This was associated with upregulated checkpoint kinase (CHK2) expression and reduced CDC2 activity (i.e., increased phosphorylation of CDC2 on Tyr-15 and decreased phosphorylation of CDC2 on Thr-161). Moreover, ß-elemene and cisplatin in combination clearly decreased the protein levels of cyclin B1 and CDC25C and increased the levels of p21(Cip1/Waf1), p27(Kip1), and GADD45 in these cells, compared with the effects of either agent alone at the same concentration. These results suggest that the ß-elemene-enhanced inhibitory effect of cisplatin on lung carcinoma cell proliferation is regulated by a CHK2-mediated CDC25C/CDC2/cyclin B1 signaling pathway and leads to the blockade of cell cycle progression at G(2)/M. A comparison of the cytotoxic efficacies of ß-elemene and three synthetic analogs (ß-elemenol, ß-elemenal, and ß-elemene fluoride) in the two lung cancer cell lines revealed that ß-elemenol and ß-elemene fluoride had the same antitumor efficacy as ß-elemene, whereas ß-elemenal was appreciably more potent than ß-elemene. Thus, although all three synthetic analogs of ß-elemene considerably suppressed NSCLC cell growth and proliferation, ß-elemenal may have greater potential as an anticancer alternative to ß-elemene in treating lung cancer and other tumors.

Differential growth inhibition and induction of apoptosis by gossypol between HCT116 and HCT116/Bax(-/-) colorectal cancer cells.

1. Bax is a very important pro-apoptosis molecule. HCT116/Bax(-/-) cells do not express the pro-apoptosis Bcl-2 family member, Bax. In the present study, the anticancer effects of gossypol on HCT116 and HCT116/Bax(-/-) cells were compared in terms of inhibition of cell growth, inhibition of colony formation and induction of apoptosis. 2. Following treatment with concentrations more than 20 micromol/L gossypol, only slight differences (not significant) were seen between HCT116 and HCT116/Bax(-/-) cells in terms of the inhibition of cell growth and induction of apoptosis. No difference was seen in the inhibition of colony formation. Gossypol had no effect at concentrations < 2 micromol/L. The only effective concentration of gossypol to result in differences between HCT116 and HCT116/Bax(-/-) cells was 5 micromol/L. However, even at this concentration, Bax deficiency did not result in complete abolition of gossypol-induced growth inhibition or apoptosis. Exposure of cells to 5 micromol/L gossypol for 24 h did not cause any significant difference in the activation of caspase 2 between HCT116 and HCT116/Bax(-/-) cells; however, activation of caspase 3, 8 and 9 was significantly elevated in HCT116 cells, with the effect on caspase 3 activation being the greatest, compared with HCT116/Bax(-/-) cells. 3. These findings suggest that the contribution of Bax to gossypol-induced growth inhibition and apoptosis is dose dependent and that gossypol-induced apoptosis requires activation of caspase 3, 8, and 9.

Gossypol induces apoptosis in human PC-3 prostate cancer cells by modulating caspase-dependent and caspase-independent cell death pathways.

The rate of gossypol-induced apoptosis does not correlate very well with the same dose of gossypol-induced cell growth inhibition, indicating an anti-proliferative effect of gossypol. Using a co-immunoprecipitation assay, it was observed that the level of Bcl-X(L) protein bound to Bax was clearly lower than that of Bcl-2 protein at 5 micro M of gossypol treatment, and the level of Bim protein bound to Bcl-X(L) was lowered at 20 micro M of gossypol treatment for 24 h, implicating that gossypol inhibits the heterodimerization of Bcl-X(L) with Bax and Bim. Gossypol-induced apoptosis is partly suppressed by as low as 0.5 micro M, but not abolished by as high as 50 micro M of a broad range caspase inhibitor, Z-VAD-FMK, suggesting that gossypol-induced apoptosis is both caspase-dependent and -independent. Furthermore, the release of apoptosis inducing factor (AIF), which triggers caspase-independent apoptosis, from mitochondria to cytosol was observed in PC-3 cells exposed to gossypol treatment. In conclusion, gossypol inhibits the proliferation and induces apoptosis in PC-3 cells. Gossypol-induced apoptosis is, at least, through inhibiting the heterodimerization of Bcl-X(L)/Bcl-2 with pro-apoptosis molecules, followed by a caspase-dependent and -independent process which involves the release of AIF from the mitochondria to cytosol.

Thalidomide down-regulates the expression of VEGF and bFGF in cisplatin-resistant human lung carcinoma cells.

Anti-angiogenic therapy represents one of the most promising treatment modalities for human cancer. Thalidomide (alpha-N-phthalimidoglutarimide) is a potent inhibitor of angiogenesis, and it is reported to overcome classical drug resistance in human multiple myeloma cells. However, the effect of this agent on the expression of angiogenic growth factors in cisplatin-resistant tumors is largely unknown. In the current study, we showed that thalidomide suppressed the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cisplatin-resistant human A549DDP lung carcinoma cells. The mRNA levels of VEGF and bFGF were markedly decreased in the A549DDP cells treated with the therapeutic concentrations of thalidomide (0.6-6 micrograms/ml), as determined by RT-PCR analysis. Consistent with these results, thalidomide also significantly reduced the protein levels of VEGF and bFGF in these cells in a dose- and time-dependent manner. This study provided evidence to support the potential therapeutic applications of thalidomide in cisplatin-resistant human lung cancer and other tumors.

Effects of thalidomide on the expression of angiogenesis growth factors in human A549 lung adenocarcinoma cells.

Angiogenesis is a critical step for the growth and metastasis of solid tumors, and it is a major therapeutic target for the development of anti-angiogenics for cancer treatment. Thalidomide is reported to be an anti-angiogenic agent, which is currently in phase II clinical trials for the treatment of advanced malignancies. However, the mechanism of thalidomide in angiogenesis is not completely understood. This study was undertaken to investigate the effect of thalidomide on the expression of angiogenesis growth factors in human lung cancer cells. In this report, we show that thalidomide downregulated the mRNA and protein expression of basic fibroblast growth factor (bFGF) in the human lung adenocarcinoma cell line A549, and that the effect of thalidomide was time and concentration-dependent. In contrast, the expression of vascular endothelial growth factor (VEGF) by thalidomide in these cells was in two phases. The mRNA and protein expression of VEGF was increased in the lung cancer cells treated with 0.6-6 microg/ml thalidomide, while higher concentrations of thalidomide decreased VEGF levels significantly in these cells. These data suggest that the anti-angiogenic or antitumor activity of thalidomide may be partly mediated through the regulation of the levels of angiogenesis growth factors.