The Impact of Extracellular Low pH on the Anti-Tumor Efficacy Against Mesothelioma

Inflammation and tumors have been demonstrated to have several common characteristics in their microenvironments. Extracellular acidosis is frequently associated both with inflammation area and tumor growth. Measurements of pH in peripheral tissues during the development of inflammation have shown extracellular pH values as low as 5.5–7.0 while the pH values of normal tissues are usually maintained at pH 7.4-7.5 mainly via pulmonary respiration and kidney perfusion of protons (Edlow & Sheldon, 1971). Similary, the extracellular pH in the central regions of tumors decreases below 6.7 in several tumors as a consequence of lactate accumulation derived from a lack of sufficient vascularization or an increase in tumor specific glycolysis under aerobic conditions combined with impaired mitochondrial oxidative phosphorylation (Simmen, 1993; Vaupel, 1989; Warburg, 1956). These pH declines affect cellular or tissue functions because their features are determined mainly by a variety of enzymatic proteins, and all enzymatic activities have each optimal pH. We have previously reported that the low pH conditions alter signal transductions. (1) The phosphorylations of several proteins were upregulated at low pH in leukemia cells (Fukamachi et al., 2001; Hirara et al., 2008). (2) CTIB, an Ikappa B beta variant, regulates cellular survival and gene expression exclusively under acidic environments in Chinese Hamster Ovary cells (Lao et al., 2005, 2006). Furthermore, the gene expressions related with tumor malignancy were upregulated at low pH in several tumor cell lines (Rofstad et al., 2006). These different characteristics dependent on extracellular pH provided us with a perspective that the inhibitory effect of anti-tumor drugs or molecular targeted inhibitors would vary at tumor-specific low pH, and the development of anti-tumor medicines, which have medical properties especially in acidic conditions, would lead to curative therapies for cancers.


Introduction
Inflammation and tumors have been demonstrated to have several common characteristics in their microenvironments.Extracellular acidosis is frequently associated both with inflammation area and tumor growth.Measurements of pH in peripheral tissues during the development of inflammation have shown extracellular pH values as low as 5.5-7.0 while the pH values of normal tissues are usually maintained at pH 7.4-7.5 mainly via pulmonary respiration and kidney perfusion of protons (Edlow & Sheldon, 1971).Similary, the extracellular pH in the central regions of tumors decreases below 6.7 in several tumors as a consequence of lactate accumulation derived from a lack of sufficient vascularization or an increase in tumor specific glycolysis under aerobic conditions combined with impaired mitochondrial oxidative phosphorylation (Simmen, 1993;Vaupel, 1989;Warburg, 1956).These pH declines affect cellular or tissue functions because their features are determined mainly by a variety of enzymatic proteins, and all enzymatic activities have each optimal pH.We have previously reported that the low pH conditions alter signal transductions.(1) The phosphorylations of several proteins were upregulated at low pH in leukemia cells (Fukamachi et al., 2001;Hirara et al., 2008).(2) CTIB, an Ikappa B beta variant, regulates cellular survival and gene expression exclusively under acidic environments in Chinese Hamster Ovary cells (Lao et al., 2005(Lao et al., , 2006)).Furthermore, the gene expressions related with tumor malignancy were upregulated at low pH in several tumor cell lines (Rofstad et al., 2006).These different characteristics dependent on extracellular pH provided us with a perspective that the inhibitory effect of anti-tumor drugs or molecular targeted inhibitors would vary at tumor-specific low pH, and the development of anti-tumor medicines, which have medical properties especially in acidic conditions, would lead to curative therapies for cancers.
Malignant mesothelioma is an aggressive tumor developed from the pleura or other mesothelioma surface.No efficient method for treatment, including chemotherapy and radiotherapy, has yet been established for advanced stage mesothelioma (Zucali & Giaccone, 2006).However, the efficacies of anti-tumor agents against not only mesothelioma but other tumors under acidic conditions have not yet been investigated exhaustively.Only some attributive information has emphasized the possibility of alteration in anti-tumor efficacy by extracellular pH and other conditions.The cytotoxicity of mitoxantrone and Table 1.Cytotoxicity of classical anti-tumor drug at different pH conditions.

Cisplatin
The cytotoxicity of cisplatin has been reported previously to show high sensitivity at low pH in EMT mouse tumor or leukemia (Laurencot & Kennedy, 1995).However, the cytotoxicity against NCI-H2052 mesothelioma was impaired at pH 6.7 as shown in Table .1 and Fig. 1.We suppose that this difference was derived from the alteration of intracellular pH value dependent on extracellular pH value and the composition of nutrients.In previous experiments, the medium pH values were set at pH 6.0 as acidic conditions in contrast to the pH value 6.7 in our reports.We confirmed that the intracellular pH values are maintained at pH above 7.1 in cells incubated in the medium with 10% FBS at pH 6.7 (data not shown).In addition to our experiments, the maintenance of intracellular pH values in weak acidic conditions was again confirmed in other tumor cell lines (Gerweck & Seethaaraman, 1996;Owen et al., 1997).Extracellular pH lower than 6.5, however, markedly reduced intracellular pH values (Rockwells, 1986).This large reduction of intracellular pH values has been associated with apoptosis being independent of the presence of anti-tumor agents, so the enhanced effect of cisplatin under acidic conditions below pH 6.0 might be derived from another mechanism.Although the pH sensitive, anti-tumor bis (aminoalcohol) dichloroplatinum(II) has been developed, but its low pH dependent cytotoxicity appeared pH only below pH 6.0 (Zorbas-Seifriends et al., 2006).The extracellular pH values in mesothelioma, are usually over 6. 7 (Jahde et al., 1992), so we presume that the cytotoxic potential of cisplatin is reduced around at pH 6.7 in mesothelioma.
The mechanism of resistance against cisplatin under low pH conditions is unknown.Cisplatin-resistance has been related with the activity of several proteins such as BRCA1, which plays multiple role in DNA repair, or p21WAF1/CIP1 proteins (Alli et al., 2011;Wei et al., 2010).Although the activity of these proteins at pH 6.7 has not been investigated, acidosis affects several protein activity (Lao et al., 2006;Rofstad et al., 2006).So the alteration of these proteins' activity may contribute to the Cisplatin-resistance at low pH.

Mitomycin C
Mitomycin C was reported to be potently its cytotoxic at low pH conditions against EMT6 tumor cells and this was associated with the upregulated binding of mitomycin C to DNA at low pH conditions (Rockwells, 1986).This result is different from our results, as shown in Table .1 and Fig. 1.The medium pH values were at pH 5.7 in previous experiments, so probably the cytotoxicity of mitomycin C would be reduced at around pH 6.7.Resistance to mitomycin C by several cancers has been reported and related with glutathion S-transferase activity (Ruiz-Gomez et al., 2000).So the quantification of the expression or activity of glutathion S-transferase under acidic conditions may be important.Although the treatment against mesothelioma using mitomycin C has been investigated, its efficacy was less than those of cisplatin (Fennell et al., 2007) or oxalipalatin (Routh et al., 2011).
In contrast, Co-treatment with mitomycin C, vinblastin and cisplatin achieved the improvement of symptoms including cough, dyspnoea and pains (Andreopoulou et al., 2004).So treatment with mitomycin C might be limited to improving the quality of life.

Doxorubicin and daunorubicin
The therapeutic effects of doxorubicin and daunorubicin against mesothelioma have been linked with the tumor-resistance mechanism of mesothelioma.Single treatment with doxorubicin and daunorubicin showed no significant anti-tumor activity (Harvey et al.,1984;Steele et al., 2001).Although this combination with cisplatin achieved some slight improvement, a high dose of doxorubicin was needed and this showed a toxic rather than curative effect (Stewart et al., 1994).These impaired efficacies of doxorubicin in mesothelioma have been thought to be due to upregulation of p-glycoprotein mediated drug efflux (Isobe et al., 1994) or multi drug associated protein (Kato et al., 1998).The expression of p-glycoprotein was up-regulated at low pH conditions in EMT6 cells and prostate cancer cells (Thews et al., 2006 ).This upregulation of p-glycoprotein is partially dependent on HIF-1 (Riganti et al., 2008) and extracellular low pH has been reported to induce HIF-1 (Mekhall et al., 2004).So, this mechanism might contribute to the pHdependent loss of cytotoxicity as shown in Fig. 1, in addition to the possibility derived from its weakly basic conformation (Raghunand et al., 1999).Drug resistance is also influenced by the activity of ROS scavengers.Most mesotheliomas express relatively large amount of manganese superoxide dismutase, catalase and cell surface NADH oxidase, and the cytotoxicitic function of ROS derived from doxorubicin were impaired in mesothelioma (Hedges et al., 2003;Kahlos et al., 1998).

Vinblastine sulfate, aclarubicin and cytochalasin D
These three agents has not been investigated at low pH conditions yet.So our experiment shown in Fig. 1 was the first to show less cytotoxicity under acidic conditions.The reduced cytotoxicity of these three drugs, however, might be explained by the pH-dependent upregulation of p-glycoprotein as well as doxorubicin because vinblastine is also a pglycoprotein substance (Gertner et al., 1998).Recent data indicated that the use of vinblastine should be limited to the improvement of symptoms as described above for mitomycin C (Andreopuoulou et al., 2004).It has been reported that the anti-tumor activity of aclarubicin is related to the production of ROS (Rogalska et al., 2008).So the mesothelioma cell, which expresses elevated amounts of ROS scavengers, may be resistant to aclarubicin.
The effects of cytochalasin D are derived from the breaking of actin filaments (Schiliwa, 1982).Human actin-depolymerizing factor and cofilin have been reported to be pH-sensitive (Pope et al., 2004).So this mechanism is likely to be associated with our results shown in Fig. 1.

The pH dependency of other anti-tumor agents
Bleomycin was reported to show pH dependency in restricted conditions.The combination of high temperature and low pH enhanced the efficacy of bleomycin and 1,3-bis(2chloroethyl)-1-nitrosourea but not methotrexate against Chinese hamster ovary, while there was no effect of pH at normal tissue temperature (Hahn & Shiu, 1983).The efficiency of bleomycin at low pH and high temperature has not been estimated against mesothelioma yet in vitro and vivo.Hyperthermia with bleomycin, however, may be worthwhile to examine against mesothelioma.
Although 5-Fluorouracil (5-FU) did not show cytotoxicity under both pH conditions as shown in Fig. 1, high dose 5-FU previously showed the potential for pH dependent mesothelioma suppression (Nissen & Tanneberg, 1981).5-FU inhibited the 3H-uridine incorporation preferentially at pH 7.4 compared to that at pH 6.8 at concentrations above 100 microM, so the comparison at each pH with higher doses of 5-FU is needed.

The effect of extracellular pH on molecular targeted inhibitors
As shown in Table 2, only four molecular targeted inhibitors, lovastatin, manumycin A, FTI-276 and cantharidin, showed higher cytotoxicity at low pH conditions in our exhaustively investigation against mesothelioma at different pH conditions using SCADs inhibitor kit.

The therapeutic capacity of statins against cancers
The pH-dependent cytotoxicity of lovastatin was observed previously in HeLa cells, mesothelioma cell line H2452 cells, pancreatic tumor cell line, BxPC-3 and Panc-1.Moreover, simvastatin, another lipophilic statin, inhibited cellular survival of HeLa cells as well as lovastatin (Fukamachi et al., 2010).These results indicated that pH-dependent cytotoxicity of lovastatin is common to several tumors and that other statins have similar properties.
Statins (3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) block the de novo synthesis of cholesterol, resulting in lower plasma cholesterol levels.Although myopathy or its more severe form rhabdomyolysis is a significant adverse effect of statin treatment, the incidence is typically less than 0.1% (Ballantyne et al., 2003).Other adverse effects including hyperplasia of the liver, squamous epithelial hyperplasia of cataracts and vascular lesions in the central nervous system have lower incidences or need extreme inhibition of the enzyme with high doses of statins (Gerson et al., 1989).Due to its active effect against cholesterol synthesis and relatively safe features, lovastatin and other statins have been prescribed for treatment of hypercholesterolaemia since the late 1980s (Tobert, 2003).Furthermore, statins might have potential for other coronary artery diseases.In patients without established cardiovascular disease but with cardiovascular risk factors, statin use was associated with significantly improved survival and large reductions in the risk of major cardiovascular events (Bruqts et al., 2009).
In addition to its great curative effect for vascular disease, the relationship of statin with cancer treatments has been discussed, including mesothelioma.Lovastatin inhibited the growth of T cell leukemia (Newman et al., 1994), pancreatic tumor (Muller et al., 1998), glioma (Jones et al., 1994), lung cancer (Maksimova et al., 2008) and prostate cancer (Hoque et al., 2008).Other statins as well as lovastatin showed cytotoxicity against tumor cell lines.Simvastatin against HT29 human colon cancer cells (Cho et al., 2008), glioma (Wu et al., 2009), pravastatin against hepatoma (Kawata et al., 1992) have been reported.Moreover, several statins reduced the migration of human pancreatic tumors and mouse melanoma (Kusama et al., 2002).In addition to the cytotoxicity and suppression of invasion against tumor cell lines, statins' curative effect against several cancers was confirmed in patients.
Conventional chemotherapy using simvastatin with irinotecan, 5-FU and leucovorin (FOLFIRI) was a feasible regimen with promising anti-tumor activity against metastatic colorectal cancers ( Lee et al., 2009).Fluvastatin reduces proliferation and increases apoptosis in women with high grade breast cancer (Garwood et al., 2010).These results indicated that statins inhibit tumor proliferation and have the potential to be applied for cancer treatments.

The mechanism of statins cytotoxicity; cholesterol and protein prenylation
The mechanism underlying statins' pH-dependent cytotoxicity is unclear.Statins inhibit HMG-CoA reductase that converts HMG-CoA to mevalonate, resulting in the reduction of farnesyl pyrophosphate, a substance of cholesterol.The important feature of cholesterol is that cholesterol is critical substance in plasma and vesicle membranes, so the inhibition of cholesterol synthesis by statins reduces mitochondrial membrane potential and induces the release of pro-apoptotic factors including cytochrome c, resulting in the apoptosis or the increase of doxorubicin sensitivity in hepatocellular carcinoma (Montero et al., 2008).The amount of cholesterol also contributed to the tumor cell migration (Sekine et al., 2010).These contribution of cholesterol to tumor movement were impaired by the reduction of cholesterol by statins (Murai et al., 2011).
Another cascade downstream of the mevalonate pathway might be important to influence cellular function or tumor survival.Farnesyl pyrophosphate is converted into geranylgeranyl pyrophosphate and both phosphates are substances of protein prenylation.
Proteins acquire lipophilicity with prenylation by farnesyl transferase or geranylgeranyl transeferase and bind to membrane or hydrophobic grooves on the surface of soluble protein factors (Gelb et al., 2006).In association with tumors, the prenylation of low molecular mass G protein has been examined, because their membrane association is induced by prenylation (Finegold et al., 1990).Lovastatin inhibits the prenylation of G proteins and alters their localization (Girgelt et al., 1994;Muller et al., 1998).Simvastatin interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA (Park et al., 2002).RhoA activity and JNK, downstream of RhoA, were also inhibited by atorvastatin, resulting in the suppression of osteosacroma invasion (Fromigue et al., 2008).In addition, Rab protein, which was recently reported to be involved in protein transport across the secretory, was geranylgeranylated by Rab geranylgeranyl transferase, and mediated cancer invasion (Leung et al., 2006).

The mechanism of statins cytotoxicity; downstream of G protein
In the downstream of these low molecular mass G protein prenylations, statins induced an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes (Paajarvi et al., 2005).Atorvastatins induce autophagy and autophagy-associated cell death in PC3 cells, likely through inhibition of geranylgeranylation (Parikh et al., 2010).GGTI-286, an inhibitor of geranylgeranyl transeferase, induced G0/G1 arrest and p21 resulting in tumor suppression (Vogt et al., 1997).These effects derived from p21 were related with the inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2 (Lin et al., 2008).Lovastatin induced U87 glioblastoma cell death in correlation with significantly increased levels of the BH3-only protein and the activation of MAPK.All of these alterations were prevented by geranylgeranyl pyrophosphate (Jiang et al., 2004).Statins have the potential to regulate gene expression related with cancer progression.MMP-9 reduction by lovastatin resulted in the suppression of invasion (Wang et al., 2000).Pitavastatin at low dose inhibits NF-kappaB activation and decreases IL-6 production induced by TNF-alpha (Wang & Kitajima, 2007).Atorvastatin inhibits inflammatory angiogenesis in mice through down-regulation of VEGF, TNF-alpha, and TGF-beta1 (Araujo et al., 2010).These reports suggest that several cancers are sensitive to statins and the inhibitors of protein prenylation.

The effect of statins against mesothelioma under acidic conditions
In contrast to statins' inhibitory effect against several cancers, little has been reported about the treatment of mesothelioma with statins or prenylation inhibitors.A few previous reports and our results, however, stress the possibility of mesothelioma treatment by statins.
Lovastatin reduced malignant mesothelioma viability with altering the membrane association of Ras, and this cytotoxic effect was impaired by addition of mevalonate (Rubins et al., 1998).It is still unclear whether the cytotoxicity was dependent on the reduction of cholesterol or on the prenylation of proteins.In regard to the doxorubicin sensitivity of mesothelioma up-regulated by statins, the inhibition of protein prenylation was thought to potentiate the cytotoxicity of doxorubicin (Riganti et al., 2006).Both mevastatin and simvastatin increased the doxorubicin sensitivity via the increase of NO, and this effect was mimicked by GGTI-286 and Y-27632, an inhibitor of ROCK which is a downstream protein of Rho GTPase.Although, there has been no discussion about the contribution of the decreased cholesterol, the decline of cholesterol level would reduce the mitochondrial membrane potential, resulting in the release of pro-apoptotic protein.So both mechanisms are thought to contribute to the up-regulation of doxorubicin sensitivity.
Our resent results indicated that mesothelioma cells can proliferate under acidic conditions, and the pH-dependent cytotoxicity was due to the pH-dependent activity of prenylated proteins or the pH-dependent alteration of protein prenylation because manumycin A, an inhibitor of farnesyl and geranylgeranyl transferase, showed pH-dependent cytotoxicity as well as statins while no pH-dependent cytotoxicity appeared with YM-53601, an inhibitor of squalene synthase (Fukamachi et al., 2010).This importance of protein prenylation was reinforced by our result that three out of the only four inhibitors including FTI-276, an inhibitor of farnesyl transferase, are related with protein prenylation as shown in Table 2.It is not clear which protein's prenylation or activity is important in mesothelioma cells under acidic conditions.The signal transductions under acidic environments are different from those at normal tissue pH.The phosphorylation of p38 was increased at low pH (Hirata et al., 2008).The activity of the Erk/Ap-1 pathway is also activated at low pH in melanoma cells (Kato et al., 2005).These results indicate that the upstream proteins of MAPK are likely to be important at low pH.In fact, the activity of ERK in cardiac myocytes at low pH was related with Ras activation (Haworth et al., 2006).The morphological changes under acidic conditions were associated with Rho kinase (Hyvelin et al., 2004).Rab11b and its mediator Rip11 regulate V-ATPase traffic elevated at low pH in duct cells (Oehike et al., 2011).These results suggested that low molecular mass G protein might be important in tumor proliferation under acidic conditions.It remains unclear yet why statins inhibit cellular proliferation preferentially in tumor growth.Recently, a novel approach to identify geranylgeranylated protein was developed using azide binding geranylgeraniol which converted to geranylgeranyl pyrophosphate in cytosol (Chan et al., 2009).So the proteins, which play a critical role for mesothelioma proliferation at low pH, will be identified soon.

The mechanism of cantharidin to suppress tumor growth
Cantharidin, a natural compound isolated from beetles, has the inhibitory activity of protein phosphatase (PP2A or PP1).Cantharidin was first found to be effective against various warts (Cusack et al., 2008).Cantharidine has also been traditionally used as an anti-cancer agent in China (Pang et al., 2007).The mechanism against cancer via the inhibition of protein phosphatase is poorly understood except for a few reports regarding to apoptosis and signal pathways.Cantharidin caused G2/M arrest through inhibition of CDK1 activity (Huang et al., 2011) and apoptosis via mitochondrial pathways including Bax, Bcl-2, Bcl-xl resulting in caspase activation (Kok et al., 2005).Cantharidin was recently shown to have passive cytotoxicity against tumor cells via the JAK/STAT pathway (Sagawa et al., 2008) and the involvement of NF-kB pathways in cantharidin-induced apoptosis was also reported (Li et al., 2011).

The problem with cantharidin and derivatives of cantharidin
Although these features of cantharidin are important for anti-tumor activity, the clinical application of cantharidin is limited due to its severe side-effects and highly toxic nature.
The protein phosphatase-inhibitory toxins have been shown to induce hyperphosphorylation of cytoskeletal proteins like keratin in isolated rat hepatocytes, and to cause disruption of the intracellular network of keratin intermediate filaments (Blankson et al., 1995).The toxins also inhibit hepatocellular processes like autophagy, endocytosis, and www.intechopen.com Mesotheliomas -Synonyms and Definition, Epidemiology, Etiology, Pathogenesis, Cyto-Histopathological Features, Clinic, Diagnosis, Treatment, Prognosis 196 protein synthesis and elicit apoptotic cell death when administered to hepatocytes in culture (Blankson et al., 2000).
Therefore, the development of more selective and effective analogs of cantharidin with less toxicity has become a challenge for cancer treatment.LB1.2, a synthesized cantharidin derivative, showed significant enhancement of cancer chemotherapy on glioblastoma and neuroblastoma cancer cells with no acute or chronic toxicity.This tumor-suppressive effect was derived from the quiescent cells cycle and caused by blocking other replication checkpoints triggered by DNA damage through the significant inhibition effect of PP2A (Rajski & Williams., 1998).Norcantharidin (NCTD) is another demethylated derivative of cantharidin possessing anti-cancer activity less toxic to normal cells, and has been used to gastric cancer (McCluskey., 2002).NCTD inhibited the activity of PP2A, and was able to promote the cell cycle from G1 to S phase with subsequent G2/M arrest (Yu et al., 2006).In addition to this phosphatase inhibitory effect, recent studies have demonstrated that cantharidin and NCTD can cause DNA damage, which may be the main contributory factor in the cytotoxicity of NCTD and cantharidin (Efferth et al., 2005).NCTD-induced caspasedependent apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential with release of cytochrome c from the mitochondria to the cytosol, and down-regulation of anti-apoptotic protein Bcl-2 (Chang et al., 2010).

The therapeutic capacity of cantharidin against mesothelioma
Combinational therapy with these new cantharidin derivatives may be used for treatment of mesothelioma.In combinational treatment with doxorubicin and LB1, the effectiveness of doxorubicin was greatly enhanced by the LB1 in the xenograft growth inhibition and lung metastases prevention of an aggressive sarcoma derived from transformed mesenchymal stem cells in syngeneic rats with little side toxicity (Zhang et al., 2010).Meanwhile, there have been several reports to show that cantharidin and its derivatives are relatively ineffective as an anti-cancer agent (Jiang et al., 1983).The contradiction may be related to cantharidin being less effective at alkaline pH but more active at acidic pH (Fukamachi et al., 2010) It remains unclear why cantharidin inhibits more strongly at acidic pH and which pathways are inhibited by cantharidin under acidic environments.The protein phosphatase, which has been related with mesothelioma proliferation, is mainly PTEN (phosphatase and tensin homologue deleted from chromosome 10).The activity of PTEN was suppressed in mesothelioma (Opitz et al., 2008), and the elevated PI3K/Akt pathway, which is the target pathway of PTEN, was repeatedly confirmed and related with mesothelioma proliferation (Altomare et al., 2005).So the inhibition of PTEN by cantharidin is not likely to lead to mesothelioma death.In other cell tumors, cantharidin induced G2/M arrest through inhibition of CDK1 (Huang et al., 2011), and induced NF-kappaB activity via constitutive phosphorylation of IKK.So the sensitivity of these proteins, especially NF-kappaB, to low pH may be important because the translocation of NF-kappaB was altered dependent on extracellular pH in T cells (unpublished data).Taken together, the pharmacologic inhibition of PP2A with less-toxic cantharidin derivatives may be a useful strategy against mesothelioma as a single treatment or combined with other anti-tumor agents including doxorubicin.

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The Impact of Extracellular Low pH on the Anti-Tumor Efficacy Against Mesothelioma 197

Aphidicolin
Aphidicolin is a specific inhibitor of DNA polymerase alpha and delta, resulting in DNA double-strand breaks leading to the activation of Ataxia-Telangiectasia Mutated: ATM (Ge & Blow, 2010), the inhibition of DNA replication and cell cycle arrest in G1/S phase (Dhillon et al., 2003).The impaired activity of aphidicolin at pH 6.7 shown in Table 2 and our previous result (Fukamachi et al., 2010) suggested that mesothelioma cells might have an acquired resistance to DNA double-strand breaks.Recently, an aphidicolin-resistant Chinese hamster V79 cell mutant had aphidicolin-resistant DNA polymerase that had an increased affinity for dNTP (Syljuasen et al., 2007).Furthermore, the checkpoint adaptation was observed in aphidicolin-treated Xenopus, and this system was expected to function in human cells (Herbst et al., 2003).The mechanism by which mesothelioma proliferates at pH 6.7 with aphidiconis is unclear yet.The most important point is that the ATM signal above has been thought to contribute to the exclusion of DNA mutated cells leading to tumorgenesis or tumor malignancy.Our result (Fig. 2) may suggest that the extracellular acidosis influences tumor malignancy via impaired ATM signals.

Bisindolmaleimide
Bisindolmaleimide is a specific inhibitor of PKC (Toullec et al., 1991).PKC is a ubiquitous phospholipid-dependent serine/threonine kinase involved in major signaling events that regulate cellular growth, migration, apoptosis, and a wide variety of biological responses to stimuli (Sukumaran & Prasadarao, 2002).Several studies have indicated that the inhibition of PKC represses tumor proliferation although it depend on the PKC class (Hu et al., 2011;Toton et al., 2011).PKCbeta1 was expressed in the majority of MPM and the treatment of MPM cell lines with PKC inhibitor showed synergy when combined with cisplatin in vitro (Faoro et al., 2008).Moreover the inhibition of protein kinase C prevents asbestos-induced c-fos and c-jun proto-oncogene expression in mesothelial cells (Fung et al., 1997).Despite these results relating tumor progression with PKC, our results showed less sensitivity to Bisindolmaleimide (Table 2 & Fig. 2).The suppressive acitivity of PKC at low pH was demonstrated in prostate carcinoma (Thews et al., 2006).So the impaired activity of Bisindolmaleimide may be due to the decline of PKC activity under acidic conditions.

PKR inhibitor
PKR, double-stranded RNA dependent protein kinase R, is activated by heme deficiency, the absence of amino acids, folded proteins accumulated in the ER, and dsRNA.The activated PKR phosphorylates eIF2α, and the phosphorylated eIF2α acts as a dominant inhibitor of the guanine exchange factor eIF2B, which prevents the recycling of eIF2 between succeeding rounds of protein synthesis and eventually leads to a global obstruction of mRNA translation initiation.This allows cells to adapt to stressful conditions by economizing on energy expended by protein synthesis (Wek et al., 2006).The adaptation process of eIF2α phoshorylation involves the selective translation of transcription factors such as activating transcription factor 4 (ATF4) (Vattem & Wek, 2004) and ATF5 (Zhou et al., 2008), which induce the expression of genes that facilitate adaptation.In cases of prolonged stress, the induction of eIF2α phosphorylation leads to cell death through the induction of apoptotic pathways (Wek et al., 2006).The results in Fig. 2 may suggest that the activity of PKR is less important in the proliferation of mesothelioma under acidic condition.Acidic conditions, in fact, enhanced the phosphorylation of eIF2, but this was confirmed at pH lower than 5.5 and not over 6.2 in a partially PKR-independent manner (Vantelon et al., 2007).So the phosphorylation of eIF2 without PKR at pH 6.7 might not cause this PKR independency at pH 6.7.Recently, there has been strong evidence to suggest that mammalian eIF2α kinases including PKR can also mediate activate glycogen synthase kinase 3 to promote the proteasomal degradation of p53 independently of eIF2α phosphorylation (Baltzis et al., 2007).So another mechanism may be critical in this reduced inhibitory effect at low pH.

4.3.4
The impaired effect of N1,N12-Diethylspermine N1,N12-Diethylspermine lost its cytotoxic activity as shown in Table 2 and Fig. 2. N1, N12 -Diethylspermine (BESpm) caused a specific induction of spermidine/spermine N'acetyltransferase (SSAT) activity, a cytosolic enzyme is induced in response to a variety of toxic agents, hormones and polyamine derivative (Casero et al., 1990).Polyamines are aliphatic cations present in all cells.SSAT is a rate-limiting step in polyamine catabolism, which catalyzes the transfer of the acetyl group from acetyl-CoA to the spermidine or spermine and has a predominant role in the regulation of intracellular polyamine concentrations in mammalian cells (Vujcic et al., 2000).Decreases in polyamines have been shown to promote decreased growth or apoptosis (Khan et al., 1992), depending on the cell type and the particular stimulus, suggesting a complex interaction between polyamines, cell growth, and cell death.Therefore, although polyamines are required for cell growth and differentiation, SSAT is thought to prevent overaccumulation of the higher polyamines from becoming toxic to the cell and may play a role in reducing the growth rate by decreasing intracellular polyamines.It has been shown that the regulation of SSAT by the natural polyamines and the anti-tumor polyamine analogues is through the polyamine response element (Wang et al., 1998).Furthermore, the superinduction of SSAT by polyamine analogues has been implicated in the cell type-specific cytotoxic response of several important human tumors including human lung cancer (Casero et al., 1990(Casero et al., , 1992;;Chang et al., 1992;Kim et al., 2005).Consistent with these previous reports, BESpm reduced cellular survival at pH 7.5 against mesothelioma as shown Fig. 2, probably via the inhibition of www.intechopen.com The Impact of Extracellular Low pH on the Anti-Tumor Efficacy Against Mesothelioma 199 SSAT by BESpm, leading to the desruption of polyamine regulation while no inhibitory effect was observed at pH 6.7.This result suggests that proliferation of mesothelioma cell line may be independent of the regulation of polyamine.

The effect of Alkylating drug
Some alkylating anti-tumor drugs including chlorambucil, carboquone and cyclophosphamide were reported to have higher cytotoxocity at low pH, probably because of their weak acid conformation or the decrease of drug resistance (Mikkelsen et al., 1985).It is difficult, however, to use them due to their causing the development of other tumors in common with alkyl agents (Rai et al., 2000).

Lowering the intracellular pH using nigericin
Low intracellular pH has been associated with enhanced cytotoxicity of anti-tumor drugs, so drugs to reduce intracellular pH have been investigated.The intracellular pH is higher than the extracellular pH, as mentioned above, so nigericin, an ionophore that acidifies the cytoplasm by exchanging cations with protons in cells placed in medium at low pH, was coincubated with anti-tumor agents.Amiloride and 4,4'-diisothiocyanostilbene 2,2-disulfonic acid, inhibitors of the Na/H and HCO3/Cl exchangers, respectively, decreased intracellular pH in the presence of nigericin at low extracellular pH against Chinese hamster ovary and human bladder cancer MGH-U1 cells (Rotin et al., 1987).

The pH-dependent cytotoxicity of N-dodecylimidazole
N-dodecylimidazole is a compound, which acquires detergent properties under acidic conditions, and is thought to be useful for selectively killing cells under intratumor low pH environments.N-dodecylimidazole displayed pH-dependent cytotoxicity against EMT-6 and MGH-U1 cells.The cytotoxicity was enhanced 100-fold at pH 6.0 compared with pH 7.0 (Boyer et al., 1993).However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents (Chen et al., 2003).Various kinds of pH sensitive detergents have recently been developed to carry the anti-tumor drugs to the tumor nest, burst, and effuse the contents, dependent on the tumor pH.An acid-cleavable PEG lipid, 1'-(4'-cholesteryloxy-3'-butenyl)-omega-methoxy-polyethylene glycolate (CVEP), has been developed that produces stable liposomes when dispersed as a minor component (0.5-5 mol %) in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).Cleavage of CVEP at mildly acidic pH results in dePEGylation of the latently fusogenic DOPE liposomes, thereby triggering the onset of content release (Boomer et al., 2009).Physical and chemical instabilities have limited the use of these drug carriers as pharmaceutical products.Recently, however, the preparation of freeze-dried pharmaceuticals has proven to be a successful strategy implemented to improve the stability of these formulations.Longcirculating and pH-sensitive liposomes containing Cisplatin are now being applied in vivo experiments (Giuberti et al., 2010).

The pH dependent cytotoxicity of tirapazamine
Low pH can substantially potentiate the cytotoxic effect of the bioreductive drug tirapazamine in HT-29 human tumor cells (Skarsgard et al., 1993).Tirapazamine is the lead member of a class of bioreductive drugs and requires metabolic activation to give a cytotoxic free radical species via a variety of cellular reductases, including NADPH cytochrome c reductase (Chinje et al., 2003).Combination therapy with tirapazamine and cisplatin has shown increased treatment efficacy compared with cisplatin alone in malignant melanoma and non-small-cell lung cancer, and also may be of benefit when combined with both radiotherapy and cisplatin in head and neck cancer (Williams et al., 2001).

Conclusion
As discussed in this review, there are no classical anti-tumor drugs which show high cytotoxicity against mesothelioma at pH 6.7.So it might be much important to prescribe both drugs whose cytotoxicity is enhanced at low pH and drugs which induce cell death at normal tissue pH for mesothelioma treatment.Although classical anti-tumor medicines have potent tumor suppressive effects, they also have severe toxicities for normal tissues.So the therapeutic regimes with lesser amounts of classical anti-tumor agents are urgently required.To this end, drugs which show higher cytotoxicity at low pH, including statins and cantharidins would be conventional candidates for co-treatment of mesothelioma.In fact, the combination treatment of solid tumors with statins enhanced anti-tumor drugs in vivo (Agarwal et al., 1999;Gao et al., 2010;O'Brien et al. 2003;Zhao et al., 2010).So the development of new compounds with anti-tumor activity preferentially at low pH would be useful for chemotherapy of mesothelioma.