|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Inostamycin prevents malignant phenotype of cancer: inhibition of phosphatidylinositol synthesis provides a therapeutic advantage for head and neck squamous cell carcinoma
Yuh Baba*†‡1, Yasumasa Kato‡1 and Kaoru Ogawa†
*Department of Otolaryngology, Tochigi National Hospital, Tochigi 320-8580, Japan, †Department of Otolaryngology, Head and Neck Surgery, Keio University, Tokyo 160-0082, Japan, and ‡Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka 238-8580, Japan
Head and neck squamous cell carcinoma is the sixth most common type of neoplasm worldwide, but its prognosis has not improved significantly in recent years. Therefore, efforts need to be intensified to gain a better understanding of this disease and develop novel treatment strategies. Inhibition of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol: inositol transferase by inostamycin, an antibiotic isolated from Streptomyces sp. MH816-AF15, induces G1 cell cycle arrest accompanied by a decrease in cyclin D1 and phosphorylated RB protein levels, along with suppression of in vitro invasive ability through reduced production of matrix metalloproteinases (MMP-2 and MMP-9) and cell motility in head and neck cancer cell lines. Furthermore, inostamycin abrogated the stimulatory effect of VEGF (vascular endothelial growth factor) on growth and migration activities of endothelial cells by targeting extracellular signal-regulated kinase-cyclin D1 and p38 pathways, respectively. Because inostamycin has both antiproliferative and anti-invasive abilities, inhibition of phosphatidylinositol synthesis could be a potent therapeutic strategy for head and neck cancer as the ‘cancer dormant therapy’, i.e. a therapeutic concept to prolong ‘time to treatment failure’ or ‘time to progression’.
Key words: cyclin D1, head and neck cancer, inostamycin, matrix metalloproteinase (MMP)-2 and -9, vascular endothelial growth factor (VEGF)
Abbreviations: CKI, CDK inhibitor, CDK, cyclin-dependent kinase, BM, basement membrane, HNSCC, head and neck squamous cell carcinoma, EC, endothelial cell, ERK, extracellular signal-regulated kinase, MMPs, matrix metalloproteinases, MAPK, mitogen-activated kinase, NF-κB, nuclear factor-κB, PI, phosphatidylinositol, pRB, Rb protein, VEGF, vascular endothelial growth factor
1Correspondence may be addressed to either of these authors (firstname.lastname@example.org or email@example.com).
HNSCC (head and neck squamous cell carcinoma) remains the sixth most common neoplasm worldwide, with ∼600000 new cases per year (Stewart and Kleihues, 2003). Recurrent and/or metastatic HNSCC patients have a poor prognosis that has remained unchanged over the past 30 years (Khuri et al., 2000; Forastiere et al., 2001). Over 50% of newly diagnosed patients with HNSCC do not achieve complete remission and relapse with metastasis to distant organs in ∼10% of cases. Therefore, more effort is needed to gain a better understanding of this disease and develop novel improved treatment strategies. Recently, inhibitors of intracellular signalling pathway or humanized antibodies for cytokine receptors to block downstream signaling pathways have been developed for the management of various cancers - the so-called ‘Molecular Targeted Therapy’. Tyrosine kinase inhibitor of epidermal growth factor receptor, which has been administered to patients with non-small-cell lung carcinoma, is in clinical trials. Unfortunately, clinical results with HNSCC have not been satisfactory for the patient, unlike cases of breast cancer treated by trastuzumab (herceptin) (Robert et al., 2006), or chronic myeloid leukemia and gastrointestinal stroma tumours treated by imatinib mesylate (Glivec, Gleevec, or STI-571; see Heinrich et al., 2003; Druker et al., 2006; de Lavallade et al., 2008). Therefore, some new targets which play a central role in the progression of HNSCC needs to be identified.
Inostamycin, a polyether compound (Figure 1), was isolated from Streptomyces sp. MH816-AF15 as a specific inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol: inositol transferase, which synthesizes PI (phosphatidylinositol) (Imoto et al., 1990). Inostamycin suppresses growth of small-cell lung carcinoma in vitro and Ehrlich tumour in vivo (Nishioka et al., 1994; Imoto et al., 1998).
A concept of extending the ‘time to treatment failure’ or ‘time to progression’ has recently been proposed, regardless of the shrinkage of tumour tissue, and implicated as the ‘cancer-dormant therapy’ (Rabinovsky et al., 2007). Based on our in vitro findings, this review highlights the inhibition of PI synthesis as a possible strategy for cancer dormant therapy in the head and neck.
Imbalance of G1 cyclin and CDK (cyclin-dependent kinase) inhibitors (CKIs) is important for tumourigenesis and tumour progression. Cyclin D1/PRAD1 acts as a positive regulator of the cell cycle via phosphorylation of pRB (Rb protein) by forming a cyclin D1-CDK4 complex (Hunter and Pines, 1994; Lees, 1995). Although pRB binds E2F, a transcription factor in its pocket releases E2F when pRB is hyperphosphorylated by CDKs, mostly having been accumulated at the G1 checkpoint.
CKIs are classified into the two following groups: members of the Ink4 family (p15, p16, p18 and p19) for cyclin D/CDK4 or cyclin D/CDK6 and the cip/kip family (p21, p27 and p57) for cyclin D/CDK4 and cyclin E/CDK2. Overexpression of cyclin D1 in HNSCC is an important prognostic marker (Nishimura et al., 1998), predicting sensitivity to chemotherapy and chemoradiotherapy (Ishiguro et al., 2003). Furthermore, imbalance between cyclin D1 and its inhibitors (p16 and p27) might be critical in the development of HNSCC (Baba et al., 1999; El-Naggar et al., 1999; Baba et al., 2001a).
Strategies to block cyclin D1 function have been studied extensively; for example, Nakashima and Clayman (2000) reported that introduction of an antisense cyclin D1 expression vector into cells reduced their in vitro growth rate and decreased tumourigenicity in athymic nude mice. Inostamycin caused G1 arrest of small-cell lung carcinoma cells accompanied by a decrease in levels of cyclin D1 and phosphorylated pRB (Imoto et al., 1998). Furthermore, we have shown that the cytostatic effect of inostamycin is not restricted to lung carcinoma cell lines, but is applicable to HNSCC cell lines with an IC50 of 0.0625-0.125 μg/ml, which are lower than those of cisplatin and 5-fluorouracil (Baba et al., 2001b; Taguchi et al., 2004).
3. Inhibition of MMP (matrix metalloproteinase) production/activity
Tumour metastasis is a complex multistep process (Fidler et al., 1978), which includes growth at the primary site, entry into the circulation (intravasation), adhesion to the basement membrane (BM) in the target organ, extravasation and growth at secondary sites. Among these steps, the intravasation and extravasation processes involve the degradation of BM by proteinases, usually the MMPs. MMP-9/gelatinase B and MMP-2/gelatinase A having specificity for type IV collagen, which acts as the backbone of BM, may play a major role in degrading the BM (Wilhelm et al., 1989; Senior et al., 1991; Morodomi et al., 1992; Bjorklund and Koivunen, 2005; Turpeenniemi-Hujanen, 2005). In HNSCC, MMP-2 and MMP-9 are associated with metastatic potential (Hong et al., 2000; Werner et al., 2002). Thus, MMP is an attractive target, and many drugs have been developed to prevent the extracellular matrix-degrading activities of MMPs in metastasis and angiogenesis. For example, marimastat and MMI-166 (Zucker et al., 2000; Katori et al., 2002) proceeded to phase III trials in patients with advanced cancer. However, these trials all failed to reach their objective of increasing survival. On the other hand, inhibition of PI synthesis affects production, but not activity, of MMP-2 and MMP-9 in HNSCC cell lines (Baba et al., 2000). Besides, inostamycin suppresses MMP production at lower doses than curcumin, which can also down-regulate MMP by inhibition of NF-κB (nuclear factor-κB) activation (Lin et al., 1998; Baba et al., 2000; Aggarwal et al., 2004; Chakravarti et al., 2006).
Angiogenesis, the formation of new blood vessels by sprouting from pre-existing capillaries or incorporating bone marrow-derived endothelial precursor cells into growing vessels, is associated with the malignant phenotype of cancer (Michi et al., 2000; Hilbe et al., 2004). In addition, it also plays a role in diverse diseases such as diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, atherosclerosis and restenosis (Cherrington et al., 2000; Hilbe et al., 2004). Clinical association of tumour vascularity with tumour aggressiveness has been clearly demonstrated in a wide variety of tumour types, including HNSCC (Sauter et al., 1999). Thus, determination of microvessel density in tumour tissue can be a useful estimate of a patient's prognosis. Inhibition of angiogenesis can repress the growth rate of tumour cells and leads to cell death due to reduced nutrition and oxygen supply to the tumour. VEGF (vascular endothelial growth factor), which plays a major role in many angiogenic processes (Michi et al., 2000; Mineta et al., 2000; Smith et al., 2000, 2001), binds to its receptor, Flk-1/KDR, to stimulate EC (endothelial cell) proliferation through the phospholipase Cγ-protein kinase C-ERK (extracellular signal-regulated kinase) pathway, but not via Ras (Takahashi et al., 1999; Shibuya, 2001). VEGF also stimulates EC migration through p38 MAPK (mitogen-activated kinase) independently of ERK (Rousseau et al., 1997). Thus, these two major MAPK pathways are good targets for reduction of angiogenesis in HNSCC. In addition, NF-κB, a transcription factor, may make a good therapeutic target to inhibit angiogenesis in HNSCC (Aggarwal et al., 2004).
Most of the clinical trials of anti-angiogenic agents were conducted in patients with advanced disease that had become resistant to conventional therapies. Phase III trials of these agents have compared the efficacy of standard chemotherapy alone and in combination with an experimental angiogenesis inhibitor. Although some studies were negative or controversial, a large number of recent clinical trials in which VEGF signalling was blocked showed significant clinical benefit. For example, an inhibitor of the Flk-1/KDR receptor (VEGF receptor), tyrosine kinase (SU11248), and a monoclonal antibody to VEGF (bevacizumab) have proved useful in clinical trials (Kabbinavar et al., 2003; Motzer et al., 2006). Inhibition of PI synthesis by inostamycin abrogated stimulation by VEGF on the growth and migration of human umbilical vein ECs through ERK-cyclin D1 and p38 pathways, respectively (Baba et al., 2004). The concentration we used was lower than that of SU11248 (Baba et al., 2004; Osusky et al., 2004).
5. Future prospects
Inostamycin can be used to inhibit PI synthesis. Although inostamycin induces apoptosis at high doses in a variety of cancer cells through protein kinase C and caspase-3 leading to H2O2 production (Imoto et al., 1998; Simizu et al., 1998a; Kawatani et al., 2000, 2003), it inhibits production of MMPs and in vitro invasion in HNSCC at low doses (Figure 2). However, there was little difference between the cytostatic and cytotoxic concentrations of inostamycin (Imoto et al., 1998; Baba et al., 2001a). Therefore, a therapeutic regimen using smaller doses is needed. A treatment strategy called ‘metronomic chemotherapy’, which involves frequent administration of chemotherapeutic agents at doses significantly below the maximum tolerated dose does exist (Gasparini, 2001), which may prove efficacious as a non-toxic strategy for inostamycin.
Although most chemotherapeutic agents and ionizing radiation induce apoptosis through activation of different caspases, in some cases, they also activate anti-apoptotic mediators such as NF-κB, leading eventually to drug resistance of tumour cells (Bharti and Aggarwal, 2002; Jackson-Bernitsas et al., 2007). Therefore, to prevent activation of the anti-apoptotic pathway, chemotherapeutic agents or γ-radiation in combination with chemo-preventive agents that can suppress anti-apoptotic mediators need to be used. Inostamycin may suppress the PI3 kinase-Akt-NF-κB pathway and potentiate the cytotoxicity of paclitaxel in small-cell lung carcinoma (Simizu et al., 1998b), possibly making it useful in combinations with other modalities such as γ-radiation and/or conventional chemotherapy.
Yuh Baba and Yasumasa Kato contributed through data collection and drafting of the manuscript. Kaoru Ogawa was involved in conception, reviewing and finally approving the version to be published. All authors read and approved the final manuscript.
We thank Dr Masaya Imoto (Keio University, Japan) for the generous gift of inostamycin, Dr Mamoru Tsukuda (Yokohama City University, Japan) for fruitful discussions, and Dr Bharat B Aggarwal (Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas MD Anderson Cancer Center) for carefully proofreading our manuscript.
This study was supported in part by a grant-in-aid from
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Received 23 October 2009; accepted 13 November 2009
Published online 14 January 2010, doi:10.1042/CBI20090310
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
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