21-Dec-2007 - BCG

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Beyond the Abstract - Cathepsin B is Involved in the Apoptosis Intrinsic Pathway Induced by Bacillus Calmette-Guérin in Transitional Cancer Cell Lines

Friday, 21 December 2007 BERKELEY, CA (UroToday.com) - This is a basic research work, where we analyzed the direct effect of BCG on transitional cancer cells (TCC) murine (MB49) and human (T24) cells lines. BCG induce apoptosis more than necrosis. It is important because its has been propose that tumor cell death by apoptosis could be a better inducer of immunity than death by necrosis.

Intravesical instillation of BCG is a standard therapy for superficial and in situ bladder carcinoma but its mechanism is not completely understood. Both immunological mechanisms and/or direct effects on tumor cells have been proposed, being the former more extensively studied. The direct effect on tumor cells has been less investigated in part because the immune therapy with BCG is administered after tumor resection, assuming that no tumor cells are present. However, it is possible that an undetectable number of tumor cells remain in the bladder. From our point of view, the biological behavior of tumor cells to BCG could contribute to the global response. For example, it has been proposed that tumor cell death by apoptosis could be a better inducer of immunity than death by necrosis (Goldszmid RS et al, J Immunol. 2003; 171: 5940-5947) and that dendritic cells loaded with apoptotic tumor cells are more efficient than dendritic cells loaded with necrotic ones in activating cytotoxic T lymphocytes (Albert ML et al, Nature. 1998; 392: 86-89). Thus, here we studied the effects of BCG on TCC lines by evaluating the participation of lysosomal hydrolase CB in apoptosis induction.

BCG induced growth inhibition in a dose-dependent manner from 0.1 mg/ml, reaching a plateau for doses higher than 2mg/ml. MB49 cells appeared to be more sensitive than T24 cells to BCG inhibition since the ID50s were 0.5mg/ml and > 2.5mg/ml respectively. The BCG concentration using here was in the therapeutic range, since 2 x 106 CFU / ml is indicate for human bladder instillation.

In agreement with other authors (Ikeda N et al, Int J Urol. 2002; 9: 29-35, Pook SH et al, J Urol. 2002; 168: 780-785), we observed that T24 and MB49 cell were capable of internalizing BCG (data no shown). In addition, it has been reported that bladder tumor cells are capable of antigen presentation and induction of an immune response upon BCG treatment (Ikeda N et al, Int J Urol. 2002; 9: 29-35). As internalization and antigenic presentation depend on lysosomal pathway, and CB is the main lysosomal hydrolase involved in MHC II antigen presentation (Deussing J et al, Proc Natl Acad Sci. USA. 1998; 95: 4516-4521), we think that CB could participate in the inhibition of cellular growth. CB protein was increased concomitantly with inhibition of cell growth after BCG treatment. An increase in the 33- and 29-27-kDa bands corresponding to the active forms of CB was detected. CB activity was also increased up to 2h after BCG addition, and CA, a specific cell permeable CB inhibitor (Buttle DJ et al, Arch Biochem Biophys. 1992, 299: 377-380) blocked not only CB activity but also reversed BCG-induced cell growth inhibition. Thus, our results show that CB participates in the inhibition of TCC growth induced by BCG.

Since induction of apoptosis by different Mycobacteria in other experimental models has been already described (Riendeau C et al, Infect Immun. 2003; 71: 254-259; Weinrauch Y et la, Annu Rev Microbiol. 1999; 53: 155-187) and that Mycobacterium Philei cell wall complex directly induces apoptosis in human bladder cancer cells (Filion MC et al, Br J Cancer. 1999; 79: 229-235), we studied whether BCG can also induce TCC apoptosis. Our results showed that BCG induced apoptosis in the three TCC lines assayed, and that CB participated in this process since it was blocked by CA.

Two initiation pathways, triggered by separate events, converge to execute apoptosis: the extrinsic one, which depends on extracellular binding to death receptors and activation of caspase 8 and the intrinsic one, which produces changes in the mitochondrial membrane potential with release of cytochrome c and activation of pro-caspase 9 (Danial N et al, Cell. 2004; 116: 205-219). It has been described that CB contributes to hepatocyte apoptosis by promoting mitochondrial release of cytochrome c (Guicciardi ME et al, J Clin Invest. 2000; 106: 1127-1137). CB has also involved in programmed death of human breast cancer cells MCF-7 by activating BID (Guicciardi ME et al, J Clin Invest. 2000; 106: 1127-1137), suggesting that CB could participate in the intrinsic pathway. To test this possibility, we evaluated whether the activation of BID and pro-caspase 9 could be detected in our TCC cell lines. Our results showed that BCG induced activation of BID and pro-caspase 9 in both T24 and MB49 cells and that this induction was mediated by CB because it could be inhibited by CA. However, some considerations should be made. First, the kinetics of BID activation is different between MB49 and T24 lines. In MB49, BID begins to disappear at 2h and is undetectable after 3h or 3.5h, while in T24, a light vanishing was detected at 1h , which was constant for at least 6h (data not shown). Besides, in MB49, truncated BID (p15) was detected at 3.5h while in T24 it was undetected at least up to 6 h post BCG addition (data no shown). This difference could be related with the fact that BCG-induced growth inhibition was higher in MB49 than in T24 lines. However, we cannot discard that it can also be due to a different sensitivity of the antibody to murine and human cell lines. Second, our results showed that the activation of pro-caspase 9 precedes that of BID. If activation of BID by CB and the subsequent activation of the mitochondrial pathway were the only mechanism, BID activation should precede pro-caspase 9 activation. Thus, other mechanisms must be involved in the activation of pro-caspase 9. Other proteins of the BID family such as Bax/Bak or other caspases could be involved in activating the mitochondrial pathway. For example, it has been reported that caspase-2 may be responsible for direct permeabilization of mitochondria, while in other instances, it may act in conjunction with Bax/Bak to amplify cytochrome c release (Enoksson M et al, J Biol Chem. 2004; 279: 49575-49578). It has recently been published that proteasome inhibitors can induce CB release, caspase -2-dependent mitochondrial permeabilization and apoptosis in human pancreatic cancer cells (Yeung BH et al, J Biol.Chem. 2006; 281: 11923-11932). Furthermore, CB could be involved in the direct activation of pro-caspase 9 or, alternatively, as described in fibrosarcoma cell apoptosis induced by TNF (Foghsgaard L et al, J cell Biology. 2001; 153: 999-1010), CB could act as a dominant executer protease inducing apoptosis of TCC.

Taken in account that apoptosis is a good inductor of immunity we can speculate that patients, whom tumor cells not undergo apoptosis after BCG therapy, could be refractory or less sensible to this immune therapy. However to arrive to this hypothesis before is necessary know the complete mechanisms through which BCG exerts its anti-tumor activity. Here, we propose that the increase in CB activity is involved in apoptosis of TCC lines induced by BCG. CB is able to activate BID and pro-caspase 9, both proteins involved in the mitochondrial apoptotic pathway. The cross talk between the extrinsic and mitochondrial apoptotic pathways as well as the participation of CB as a dominant executer protease awaits further investigation. To our knowledge, this is the first report showing that CB is involved in TCC apoptosis.

Written by Eduardo O. Sandes MD, as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

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