Design,synthesis and antitumor activity of a novel PEG-A6-conjugated irinotecan derivative

A novel PEG-A6-conjugated irinotecan derivative 8 was designed and synthesized as antitumor agent by the PEGylation and A6-peptide modification of irinotecan. In vivo antitumor activity screening assay revealed that 8 exhibited better in vivo antiproliferation activity than irinotecan and its previous PEG-cRGD-conjugated derivative BGC0222 in MIA PaCa-2, NCI-H446, MDA-MB-231, HT-29 and NCI-N87 xenograft models, while the tumor of one in six mice in NCI-H446 assay and the tumors of two in six mice in MIA PaCa-2 assay completely subsided and disappeared within the 21-day period of 8-treatment, indicating that 8 should be a potential antitumor agent.     

Inhibition of Mcl-1 promotes senescence in cancer cells: implications for preventing tumor growth and chemotherapy resistance

Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53⁺ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53⁻ cells restored CIS to similar levels as p53⁺ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53⁺ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Suppression of tumor angiogenesis by targeting the protein neddylation pathway

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin–RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.     

Anti-Cancer Effect of HIV-1 Viral Protein R on Doxorubicin Resistant Neuroblastoma

Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX). To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.     

Wentilactone A as a novel potential antitumor agent induces apoptosis and G2/M arrest of human lung carcinoma cells,and is mediated by HRas-GTP accumulation to excessively activate the Ras/Raf/ERK/p53-p21 pathway

Chemotherapy remains the common therapeutic for patients with lung cancer. Novel, selective antitumor agents are pressingly needed. This study is the first to investigate a different, however, effective antitumor drug candidate Wentilactone A (WA) for its development as a novel agent. In NCI-H460 and NCI-H446 cell lines, WA triggered G2/M phase arrest and mitochondrial-related apoptosis, accompanying the accumulation of reactive oxygen species (ROS). It also induced activation of mitogen-activated protein kinase and p53 and increased expression of p21. When we pre-treated cells with ERK, JNK, p38, p53 inhibitor or NAC followed by WA treatment, only ERK and p53 inhibitors blocked WA-induced apoptosis and G2/M arrest. We further observed Ras (HRas, KRas and NRas) and Raf activation, and found that WA treatment increased HRas–Raf activation. Knockdown of HRas by using small interfering RNA (siRNA) abolished WA-induced apoptosis and G2/M arrest. HRas siRNA also halted Raf, ERK, p53 activation and p21 accumulation. Molecular docking analysis suggested that WA could bind to HRas-GTP, causing accumulation of Ras-GTP and excessive activation of Raf/ERK/p53-p21. The direct binding affinity was confirmed by surface plasmon resonance (SPR). In vivo, WA suppressed tumor growth without adverse toxicity and presented the same mechanism as that in vitro. Taken together, these findings suggest WA as a promising novel, potent and selective antitumor drug candidate for lung cancer.     

Study on antitumor activities of the chrysin-chromene-spirooxindole on Lewis lung carcinoma C57BL/6 mice in vivo

The our previous study synthesized the chrysin-chromene-spirooxindole hybrids 3, and further found compound 3e had good antitumor activity against A549 cells in vitro through multi-target co-regulation of the p53 signalling pathway to inhibit the proliferation of A549 cells. This study was designed to evaluate the antitumor effects of compound 3e on Lewis lung carcinoma of C57BL/6 mice in vivo. Compound 3e significantly inhibited the growth of transplanted tumors in C57BL/6 mice and induced the apoptosis of tumor cells. Further studies showed that compound 3e activates and expands the anti-cancer activity of p53 by inhibiting the expression of MDM2, Akt and 5-Lox proteins, accordingly promotes the expressions Bax and inhibit the Bcl-2 protein, the release of Cyt c as well, which resulted in the activation of apoptotic pathway in tumor cells eventually. Moreover, Compound 3e inhibited tumor metastasis by down-regulating VEGF, ICAM-1 and MMP-2 protein expression and angiogenesis. These results suggested that compound 3e exerts an effective antitumor activity in vivo through activating the p53 signaling pathway, which could be exploited as a promising candidate for the development of new anti-tumour drugs.     

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O⁶ methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O⁶-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21^cip1/waf1. We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21^cip1/waf1 and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.     

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.Methods and resultsBy cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.ConclusionsSLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.General SignificanceThis work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.     

Antitumor Activity of a 5-Hydroxy-1H-Pyrrol-2-(5H)-One-Based Synthetic Small Molecule In Vitro and In Vivo

Alternative chemo-reagents are in great demand because chemotherapy resistance is one of the major challenges in current cancer treatment. 5-hydoxy-1H-pyrrol-2-(5H)-one is an important N-heterocyclic scaffold that is present in natural products and medicinal chemistry. However, its antitumor activity has not been systematically explored. In this study, we screened a panel of 5-hydoxy-1H-pyrrol-2-(5H)-one derivatives and identified compound 1d as possessing strong anti-proliferative activity in multiple cancer cell lines. Cell cycle analysis revealed that 1d can induce S-phase cell cycle arrest and that HCT116 was sensitive to 1d-induced apoptosis. Further analysis indicated that 1d preferentially induced DNA damage and p53 activation in HCT116 cells and that 1d-induced apoptosis is partly dependent on p53. Furthermore, we showed that 1d significantly suppressed tumor growth in xenograft tumor models in vivo. Taken together, our results suggest that 5-hydoxy-1H-pyrrol-2-(5H)-one derivatives bear potential antitumor activity and that 1d is an effective agent for cancer treatment.     

FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions.     

Anti-cancer effect of marchantin C via inducing lung cancer cellular senescence associated with less secretory phenotype

BackgroundLung cancer is the leading cause of global cancer deaths. Current chemotherapeutic agents for lung cancer treatment are generally accompanied with severe side effects. Here, we report that marchantin C (Mar-C), a potential natural compound with little chemotherapeutic toxicity, exerts a well anti-tumor effect against lung cancer via inducing cellular senescence.MethodsThe antitumor activity of Mar-C was evaluated by MTT and colony formation in vitro cytotoxicity assays, and xenograft and homograft in vivo model. Antitumor mechanisms of Mar-C were investigated through SA-β-gal staining, Q-PCR, immunoblotting, immunofluorescence, protein array and siRNA knocking-down analysis.ResultsMar-C selectively induces senescence of lung cancer cells with limited cytotoxicity on normal or non-neoplastic cells. Mar-C-induced senescence was associated with the elevation of ROS and activation of DNA-damage, and largely dependent of prolonged p21^CIP1 accumulation. The senescence-associated secretory phenotype (SASP) induced by Mar-C was distinct from doxorubicin-induced. Furthermore, Mar-C exhibited an inhibitory activity on tumor growth with little toxicity in animal studies, and significantly prolonged the survival time of tumor-bearing mice than that of doxorubicin or vehicle treatments.ConclusionMar-C selectively inhibited tumor growth via the induction of cancer cell senescence and had little chemotherapeutic toxicity, suggesting the potential of Mar-C as a promising anticancer agent.General significanceThis study provided evidence to identify a novelty of Mar-C that exerted antitumor activity on lung cancer through induction of senescence with limited toxicity.     

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2–p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26–HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.     

Regorafenib: Antitumor Activity upon Mono and Combination Therapy in Preclinical Pediatric Malignancy Models

The multikinase inhibitor regorafenib (BAY 73–4506) exerts both anti-angiogenic and anti-tumorigenic activity in adult solid malignancies mainly advanced colorectal cancer and gastrointestinal stromal tumors. We intended to explore preclinically the potential of regorafenib against solid pediatric malignancies alone and in combination with anticancer agents to guide the pediatric development plan. In vitro effects on cell proliferation were screened against 33 solid tumor cell lines of the Innovative Therapies for Children with Cancer (ITCC) panel covering five pediatric solid malignancies. Regorafenib inhibited cell proliferation with a mean half maximal growth inhibition of 12.5 μmol/L (range 0.7 μmol/L to 28 μmol/L). In vivo, regorafenib was evaluated alone at 10 or 30 mg/kg/d or in combination with radiation, irinotecan or the mitogen-activated protein kinase kinase (MEK) inhibitor refametinib against various tumor types, including patient-derived brain tumor models with an amplified platelet-derived growth factor receptor A (PDGFRA) gene. Regorafenib alone significantly inhibited tumor growth in all xenografts derived from nervous system and connective tissue tumors. Enhanced effects were observed when regorafenib was combined with irradiation and irinotecan against PDGFRA amplified IGRG93 glioma and IGRM57 medulloblastoma respectively, resulting in 100% tumor regressions. Antitumor activity was associated with decreased tumor vascularization, inhibition of PDGFR signaling, and induction of apoptotic cell death. Our work demonstrates that regorafenib exhibits significant antitumor activity in a wide spectrum of preclinical pediatric models through inhibition of angiogenesis and induction of apoptosis. Furthermore, radio- and chemosensitizing effects were observed with DNA damaging agents in PDGFR amplified tumors.