Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Doxorubicin induces the cleavage of Poly (ADP-ribose) polymerase, a marker of programmed cell death (apoptosis) in mouse cardiomyocytes

Cancer is one of the leading causes of death in the world. Despite this, a growing number of people are surviving the disease due to medical advancements and the development of numerous new therapies. Doxorubicin, a chemotherapeutic agent, is a widely-used and successful first-line anti-tumour treatment. However, the established toxic and deleterious effects of the drug on the cardiovascular system confer increased risk of congestive heart failure, thereby necessitating the use of reduced doxorubicin doses. In order to investigate how these events are initiated, mouse cardiomyocytes (HL-1) were treated in vitro with varying concentrations of doxorubicin (0.5-4.0 µmol/L). Following treatment (24h), a marked level of cell death was observed in comparison to untreated cardiomyocytes; the level of death appeared to correlate with the concentration of the drug used. Western blotting revealed the cleavage of full length Poly (ADP-ribose) polymerase (PARP) into 89 and 24kDa fragments, a process which is instrumental in triggering programmed cell death/apoptosis. Importantly, results suggested that this event may be independent of caspase 3 cleavage and thus activation. A number of previous studies have reported a functional role for both Mitofusin-2 (Mfn2) and NADPH oxidase 2 (Nox2) in the cardiotoxic response. Given that PARP cleavage is a validated indicator of cellular apoptosis, these results clearly indicate that this marker could be used in future studies when determining if depletion of the above proteins would cause a reduction in or eradicate the pro-apoptotic action of this agent on cardiomyocytes. Such investigations may lead to significant developments in ensuring that doxorubicin can achieve its full therapeutic anti-tumour potential without causing the subsequent deleterious effects on the cardiovascular system.

The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B

Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.

Rheumatologists' judgements about the efficacy of anti-TNF therapy in two neighbouring regions

Objectives: The requirement in Northern Ireland to prescribe biologic agents according to NICE/BSR guidelines and within a fixed budget has created a waiting list for treatment that has no parallel in the Republic of Ireland. The study investigated the bearing this situation may have on had on the consultants’ judgements in the respective areas.

Methods: 78 case vignettes created from the data on real patients with RA treated with biologics in the north and south of Ireland were appraised by 9 southern and 8 northern consultants who judged the clinical benefit and significance of the patients’ condition after a trial of therapy. Quantitative (Clinical Judgement Analysis) and Qualitative (Focus groups) techniques were used.

Results: Northern consultants perceived a slightly greater degree of clinical benefit after a trial of therapy than southern consultants. Judgment models of northern and southern consultants were broadly comparable. The latter tended to be more uniform in their judgments than the southern group. Focus group discussions with consultants largely validated the findings of the quantitative analysis but revealed how clinical judgment analysis might be misled by gaming strategies.

Conclusions: Despite the absence of overt rationing in the south of Ireland, as far as the judgment of therapeutic benefit from biologics was concerned, the clinical judgment policies of practitioners were very similar to those in the north. The adoption of NICE/BSR guidelines in the north may have improved the uniformity of clinical practice in Northern Ireland.