Guy Makin

Apoptosis and cancer chemotherapy

Abstract Apoptosis is a fundamental mechanism of cell death that can be engaged by a range of cellular insults. One of the major modes of action of chemotherapeutic drugs may be via the activation of apoptosis. Understanding how the cell death program is engaged following an insult, and hence why it fails to be engaged in certain settings, offers a novel approach to overcoming the clinical problem of drug resistance. The tumour suppressor gene p53 and its downstream effector genes p21, mdm-2,and gadd45 seem to be important in the cellular response to genotoxic drug induced damage. Considerable evidence has accrued about the effect of mutations of this pathway on drug sensitivity and this is discussed.The expanding Bcl-2 family of proteins also play an important role in the cell death program. Evidence suggests that these proteins may function as integrators of damage signals, and may be the final decision point as to whether a cell lives or dies. These proteins may thus represent a logical target for new approaches to overcoming drug resistance.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro‐apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH‐EP1 and SH‐SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N‐terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N‐terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH‐SY5Y) or lost (SH‐EP1) clonogenic survival after taxol treatment. Suppression of taxol‐induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH‐SY5Y cells. The data suggest that a further drug damage‐induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol‐resistant cells.

Chronic hypoxia promotes hypoxia-inducible factor-1alpha-dependent resistance to etoposide and vincristine in neuroblastoma cells.

Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature. Direct measurement of low oxygen levels in a range of adult tumor types has correlated tumor hypoxia with advanced stage, poor response to chemotherapy and radiotherapy, and poor prognosis. Little is known about the importance of hypoxia in pediatric tumors; therefore, we evaluated the effects of hypoxia on the response of the neuroblastoma cell lines SH-EP1 and SH-SY5Y to the clinically relevant drugs, vincristine, etoposide, and cisplatin. Short periods of hypoxia (1% O2) of up to 16 hours had no effect on drug-induced apoptosis or clonogenic survival. Prolonged hypoxia of 1 to 7 days leads to reduction in vincristine- and etoposide-induced apoptosis in SH-SY5Y and SH-EP1 cells, and this was reflected in increased clonogenic survival under these conditions. Neither short-term nor prolonged hypoxia had any effect on the clonogenic response to cisplatin in SH-SY5Y cells. Hypoxia-inducible factor-1 (HIF-1) α was stabilized in these cell lines within 2 hours of hypoxia but was no longer detectable beyond 48 hours of hypoxia. Up-regulation of carbonic anhydrase IX showed HIF-1α to be transcriptionally active. Down-regulation of HIF-1α by short hairpin RNA interference and the small-molecule 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole reduced hypoxia-induced drug resistance. These results suggest that prolonged hypoxia leads to resistance to clinically relevant drugs in neuroblastoma and that therapies aimed at inhibiting HIF-1α function may be useful in overcoming drug resistance in this tumor. [Mol Cancer Ther 2006;5(9):2241–50]

Leptomeningeal melanoma in childhood

Malignant melanoma (MM) is one of the least common types of childhood cancer, accounting for less than 1% of all pediatric malignancies. Neurocutaneous melanosis (NCM) is a rare phakomatosis consisting of congenital abnormal pigmentation of the skin and meninges. The meningeal lesions are particularly prone to malignant change.

Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy?

Drug resistance is a fundamental problem in the treatment of most common human cancers. Our understanding of the cellular mechanisms underlying death and survival has allowed the development of rational approaches to overcoming drug resistance. The mitogen activated protein kinase family of protein serine/threonine kinases has been implicated in this complex web of signalling, with some members acting to enhance death and other members to prevent it. A recent publication by MacKeigan et al is the first to demonstrate an enhancement of drug-induced cell death by simultaneous blockade of MEK-mediated survival signalling, and offers the potential for targeted adjuvant therapy as a means of overcoming drug resistance.

Targeting apoptosis in cancer chemotherapy

The aim of cancer biology is for a better understanding of the molecular basis of cancer, with the expectation that this will result in therapeutic advances and improved outcomes for patients. The discovery of apoptosis has contributed much to our understanding of the mechanisms of cell death, in both normal and neoplastic cells, and it has led to changes in the way that chemotherapy has been viewed. It is now increasingly accepted that part of the efficacy of conventional chemotherapeutic drugs is due to their ability to induce apoptosis, although this area is not without controversy. This has allowed advances in the fundamental understanding of apoptosis to have similar impacts upon cancer biology. It is now possible to construct a framework where cellular decisions about life and death can be seen as the result of a balance of pro- and anti-apoptotic signals, enacted by protein members of the Bcl-2 family, controlling mitochondrial cytochrome c release. This framework has allowed the importance of providing death signals and abrogating survival signals to both be appreciated. A range of novel approaches to the induction of apoptosis by downregulating survival signalling are described. In addition, many alternative strategies aimed at targeting particular molecular abnormalities of neoplastic cells as a means of inducing apoptosis are also under investigation and several of these are discussed. The mechanistic understanding of cell death will have profound impacts upon the practice of oncology and outlook for many patients.

Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing’s sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1). In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K) pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target to reverse drug resistance in paediatric osteosarcoma. The novel finding of HIF-1α independent drug resistance suggests however other hypoxia related targets may be more relevant in paediatric osteosarcoma.

Alteration of cellular behavior and response to PI3K pathway inhibition by culture in 3D collagen gels.

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.

Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737–induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors.

A small molecule inhibitor of XIAP induces apoptosis and synergises with vinorelbine and cisplatin in NSCLC.

Background:Evasion of apoptosis contributes to the pathogenesis of solid tumours including non-small cell lung cancer (NSCLC). Malignant cells resist apoptosis through over-expression of inhibitor of apoptosis proteins (IAPs), such as X-linked IAP (XIAP).Methods:A phenylurea-based small molecule inhibitor of XIAP, XIAP antagonist compound (XAC) 1396-11, was investigated preclincally to determine its ability to sensitise to clinically relevant cytotoxics, potentially allowing dose reduction while maintaining therapeutic efficacy.Results:XIAP protein expression was detected in six NSCLC cell lines examined. The cytotoxicity of XAC 1396-11 against cultured NSCLC cell lines in vitro was concentration- and time-dependent in both short-term and clonogenic assays. XAC 1396-11-induced apoptosis was confirmed by PARP cleavage and characteristic nuclear morphology. XAC 1396-11 synergised with vinorelbine±cisplatin in H460 and A549 NSCLC cells. The mechanism of synergy was enhanced apoptosis, shown by increased cleavage of caspase-3 and PARP and by the reversal of synergy by a pan-caspase inhibitor. Synergy between XAC 1396-11 and vinorelbine was augmented by optimising drug scheduling with superior effects when XAC 1396-11 was administered before vinorelbine.Conclusion:These preclinical data suggest that XIAP inhibition in combination with vinorelbine holds potential as a therapeutic strategy in NSCLC.