Organometallic iridium(III) anticancer complexes with new mechanisms of action: NCI-60 screening, mitochondrial targeting, and apoptosis

Platinum complexes related to cisplatin, cis-[PtCl2(NH3)2], are successful anticancer drugs; however, other transition metal complexes offer potential for combating cisplatin resistance, decreasing side effects, and widening the spectrum of activity. Organometallic half-sandwich iridium (IrIII) complexes [Ir(Cpx)(XY)Cl]+/0 (Cpx = biphenyltetramethylcyclopentadienyl and XY = phenanthroline (1), bipyridine (2), or phenylpyridine (3)) all hydrolyze rapidly, forming monofunctional G adducts on DNA with additional intercalation of the phenyl substituents on the Cpx ring. In comparison, highly potent complex 4 (Cpx = phenyltetramethylcyclopentadienyl and XY = N,N-dimethylphenylazopyridine) does not hydrolyze. All show higher potency toward A2780 human ovarian cancer cells compared to cisplatin, with 1, 3, and 4 also demonstrating higher potency in the National Cancer Institute (NCI) NCI-60 cell-line screen. Use of the NCI COMPARE algorithm (which predicts mechanisms of action (MoAs) for emerging anticancer compounds by correlating NCI-60 patterns of sensitivity) shows that the MoA of these IrIII complexes has no correlation to cisplatin (or oxaliplatin), with 3 and 4 emerging as particularly novel compounds. Those findings by COMPARE were experimentally probed by transmission electron microscopy (TEM) of A2780 cells exposed to 1, showing mitochondrial swelling and activation of apoptosis after 24 h. Significant changes in mitochondrial membrane polarization were detected by flow cytometry, and the potency of the complexes was enhanced ca. 5× by co-administration with a low concentration (5 μM) of the γ-glutamyl cysteine synthetase inhibitor L-buthionine sulfoximine (L-BSO). These studies reveal potential polypharmacology of organometallic IrIII complexes, with MoA and cell selectivity governed by structural changes in the chelating ligands

Upregulation of p27 and its inhibition of CDK2/cyclin E activity following DNA damage by a novel platinum agent are dependent on the expression of p21

The cisplatin analogue 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinumIV (DAP) is a DNA-damaging agent that will be entering clinical trials for its potent cytotoxic effects against cisplatin-resistant tumour cells. This cytotoxicity may reside in its ability to selectively activate G1-phase checkpoint response by inhibiting CDKs via the p53/p21 pathway. We have now evaluated the role of another CDK inhibitor p27 as a contributor to DAP-mediated inhibition of G1-phase CDK2 activity. Our studies in ovarian A2780 tumour cells demonstrate that p27 levels induced by DAP are comparable to or greater than those seen for p21. The induction of p27 is not through a transcriptional mechanism, but rather is due to a four-fold increase in protein stabilisation through a mechanism dependent on p21. Moreover, DAP-induced p21 promoted the selective increase of p27 in the CDK2 complex, but not in CDK4 complex, and this selective increase contributed to inhibition of the CDK2 kinase activity. The inhibited complex contained either p27 or p21, but not both, with the relative levels of cyclin E associated with p27 and p21 indicating that about 25% of the inhibition of CDK2 activity was due to p27 and 75% due to p21. This study provides the first evidence that p27 upregulation is directly attributable to activation of the p53/p21 pathway by a DNA-damaging agent, and promulgates p53/p21/p27 axis as a significant component of checkpoint response

Pharmacological Effects of Asiatic acid in Glioblastoma Cells under Hypoxia

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Despite current treatment options including surgery followed by radiation and chemotherapy with temozolomide (TMZ) and cisplatin, the median survival rate remains low (<16 months). Combined with increasing drug resistance and the inability of some compounds to cross the blood brain barrier (BBB), novel compounds are being sought for the treatment of this disease. Here, we aimed to examine the pharmacological effect of Asiatic acid (AA) in glioblastoma under hypoxia. To investigate the effects of AA on cell viability, proliferation, apoptosis and wound healing, SVG p12 fetal glia and U87-MG grade IV glioblastoma cells were cultured under normoxic (21% O2) and hypoxic (1% O2) conditions. In normoxia, AA reduced cell viability in U87-MG cells in a time and concentration-dependent manner. A significant decrease in viability, compared to cisplatin, was observed following 2hrs of AA treatment with no significant changes in cell proliferation or cell cycle progression observed. Under hypoxia, a significantly greater number of cells underwent apoptosis in comparison to cisplatin. While cisplatin showed a reduction in wound healing in normoxia, a significantly greater reduction was observed following AA treatment. An overall reduction in wound healing was observed under hypoxia. The results of this study show that AA has cytotoxic effects on glioma cell lines and has the potential to become an alternative treatment for glioblastoma

Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin

Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined.Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect.Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment.Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to

MicroRNA-mediated drug resistance in breast cancer

Chemoresistance is one of the major hurdles to overcome for the successful treatment of breast cancer. At present, there are several mechanisms proposed to explain drug resistance to chemotherapeutic agents, including decreased intracellular drug concentrations, mediated by drug transporters and metabolic enzymes; impaired cellular responses that affect cell cycle arrest, apoptosis, and DNA repair; the induction of signaling pathways that promote the progression of cancer cell populations; perturbations in DNA methylation and histone modifications; and alterations in the availability of drug targets. Both genetic and epigenetic theories have been put forward to explain the mechanisms of drug resistance. Recently, a small non-coding class of RNAs, known as microRNAs, has been identified as master regulators of key genes implicated in mechanisms of chemoresistance. This article reviews the role of microRNAs in regulating chemoresistance and highlights potential therapeutic targets for reversing miRNA-mediated drug resistance. In the future, microRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of drug-resistant breast cancers

Global Analysis of DNA Methylation by Methyl-Capture Sequencing Reveals Epigenetic Control of Cisplatin Resistance in Ovarian Cancer Cell

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1

Dietary tuna hydrolysate modulates growth performance, immune response, intestinal morphology and resistance to Streptococcus iniae in juvenile barramundi, Lates calcarifer

This study investigated the effects of tuna hydrolysate (TH) inclusion in fishmeal (FM) based diets on the growth performance, innate immune response, intestinal health and resistance to Streptococcus iniae infection in juvenile barramundi, Lates calcarifer. Five isonitrogenous and isoenergetic experimental diets were prepared with TH, replacing FM at levels of 0% (control) 5%, 10%, 15% and 20%, and fed fish to apparent satiation three times daily for 8 weeks. The results showed that fish fed diets containing 5% and 10% TH had significantly higher final body weight and specific growth rate than the control. A significant reduction in blood glucose was found in fish fed 10%, 15% and 20% TH compared to those in the control whereas none of the other measured blood and serum indices were influenced by TH inclusion. Histological observation revealed a significant enhancement in goblet cell numbers in distal intestine of fish fed 5 to 10% TH in the diet. Moreover, fish fed 10% TH exhibited the highest resistance against Streptococcus iniae infection during a bacterial challenge trial. These findings therefore demonstrate that the replacement of 5 to 10% FM with TH improves growth, immune response, intestinal health and disease resistance in juvenile barramundi