Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway

Malignant tumors are one of the main scourges of humanity. In 2012 alone, about 8.2 million people died of carcinoma worldwide, and—according to the World Health Organization—[1] this figure is expected to rise to 13 million over the next 20 years.

From the molecular biology point of view, cancer is defined as any type of malignant neoplasm, independent of the organ or the tissue from which the tumor originates [2]. Cancer develops when the normal balance between mitosis and apoptosis is disrupted [3]. The subsequent increase in the proliferation rate or the decrease in the apoptosis rate of cells results in uncontrolled cellular growth [4, 5]. Thus, the current aim of cancer research is to increase the apoptosis rate in tumor cells—while simultaneously limiting concurrent death of healthy cells—and to induce controlled apoptosis in abnormal cells [6].

Therefore, comprehensive knowledge of the mechanisms of apoptosis in the different types of cells is vital for developing potential cancer therapies [7]. The term apoptosis refers to programmed cell death, which constitutes an important mechanism for maintaining tissue homeostasis [8]. The characteristic feature in this respect is that apoptosis is actively induced by the respective cell itself; thus, the cell is part of the metabolism. Activation of proteolytic enzymes termed caspases results in cellular shrinkage, condensation, and fragmentation of the cell nucleus, loss of cellular adhesion, and finally in apoptosis [9]. Apoptosis is induced by activation of important signal-transduction cascades [10]. Here, extrinsic and intrinsic signaling pathways have to be differentiated [11]. After the binding of a ligand to the death receptor of the tumor necrosis factor receptor family CD 95 (APO-1/Fas) or TRAIL receptors, caspase-8 is activated via the extrinsic signaling pathway [12, 13]. In turn, caspase-8 activates caspase-3 (effector caspase) [14], thus inducing apoptosis. The intrinsic signaling pathway is the cellular response to stress [15]. Changes in the mitochondrial membrane result in the collapse of its potential and thus in the release of cytochrome C, which subsequently irreversibly instigates the entire caspase cascade by activating caspase-9 [16] (Fig. 1).

A highly potent inductor of apoptosis is staurosporine, an alkaloid that inhibits many different kinases [17]. Several new derivatives of this substance, which was originally isolated from Streptomyces staurosporeus, have been synthesized in clinical trials to be used in anticancer therapies [18]. So far, many different kinase pathways of staurosporine-induced apoptosis have been discussed for various tumor entities [19‐21].

The aim of this study was to identify the effect of staurosporine in pancreatic and colorectal carcinoma cells and its apoptosis-inducing signaling pathway.

Apoptosis is a key regulator of physiological growth control and tissue homeostasis. One of the most important findings in cancer research of the past few years is that apoptosis has a high impact on the development of tumors as well as on their response to chemotherapy [22, 23]. Bcl2 proteins residing on the mitochondrial membrane serve a regulatory function in the intrinsic apoptotic signaling pathway. They strictly control this pathway by inducing mitochondrial outer membrane permeabilization (MOMP). All proteins of the family show similar structural domains, called “Bcl2 homology (BH) domains” (named BH1, BH2, BH3, and BH4). They can be divided into three subgroups: the prosurvival members (Bcl2, Bcl-xl, Bcl-w, MCL-1, and A1), the pro-apoptotic members, which include the MOMP effectors (mainly BAX and BAK), and the BH3-only proteins, so called because they have only the BH3 domain (Bad, BIM, BID, PUMA, NOXA) [24].

Pro-apoptotic factors such as BAX, BAK, and Bad and the anti-apoptotic factors Bcl2 and Bcl-xl are delicately balanced, and this balance is often lacking in tumor cells [25]. For instance, dysfunction of BAX may further the tumor genesis of cells; thus, many chemotherapies try to indirectly intervene in this process [26]. Derivatives such as oblimersen sodium, AT-101, ABT-263, and GX15-070 are currently under clinical investigation [27, 28]. Furthermore, kinase inhibitors are also constantly used in cancer treatment [29, 30].

A highly potent inductor of apoptosis is staurosporine, an alkaloid that inhibits many different kinases [17]. In 2001, Stepczynska et al. [18] showed that staurosporine—as a broadband kinase inhibitor—induced apoptosis in Jurkat cells resistant to chemotherapeutic agents. Chae et al. [31] reported on the apoptosis-inducing effect of staurosporine in osteoblasts and Xue et al. [32] on this effect in breast carcinoma cells. The inhibiting effect of staurosporine on cell adhesion, mobilization, and invasion could also be shown in lung carcinoma cells [33]. To different degrees, staurosporine also seems to affect the induction of apoptosis in pancreatic and colorectal carcinoma cells. However, the signaling pathway by which staurosporine induces apoptosis has yet remained unclear.

The current study shows that—in the pancreatic and colorectal cancer cell lines Panc-1, PaTu 8988t, and SW 480—staurosporine does not influence BAX, but influences the anti-apoptotic factor Bcl2. The Bcl2 protein is often overexpressed in several types of cancer, for instance in breast, lung, and ovarian cancer or in malignant melanoma. Therefore, evidence of Bcl2 in cells is often associated with unfavorable outcome [34].

Bcl2 is expressed in the pancreatic cancer cell line PaTu 8988t. If Bcl2 is produced, apoptosis may be induced by suppressive medication. For this reason, the effect of BH3-mimetica has been repeatedly investigated in clinical trials, because these drugs induce apoptosis by binding and inhibiting anti-apoptotic members of the Bcl2 protein family [35, 36].

However, Bcl2 expression decreased after the administration of staurosporine in PaTu 8988t carcinoma cells. But why does staurosporine, as a kinase inhibitor, cause activation of the intrinsic apoptotic pathway? A possible explanatory approach could be that the imbalance between BAX and Bcl2—thus between pro-apoptotic and anti-apoptotic factors—probably induces apoptosis. This hypothesis is supported by the activation of full length caspase-9, shifting the balance to cleaved caspase-9. Cell apoptosis is eventually induced by activation of the intrinsic signaling pathway via caspase-9.

Bad—a further important protein in the apoptosis process—stands for Bcl2 antagonist of cell death. This protein may develop pro-apoptotic effects due to the heterodimerization with anti-apoptotic factors, for instance Bcl2, by binding to and thus blocking Bcl2 [37]. After stimulation with staurosporine, expression of Bad cannot any longer be detected by Western blot analysis. A possible explanation may be heterodimerization of Bad and Bcl2 that further shifts the balance between pro-apoptotic and anti-apoptotic factors in favor of pro-apoptotic factors. But degradation pathways at the level of transcription, translation level, or protein modification/clearance would also be conceivable.

Yuste et al. investigated whether overexpression of Bcl2 can rescue cells from staurosporine-induced apoptosis. They found that overexpression of Bcl2 increased the resistance of cells to staurosporine up to 1 µM. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated, and cells died by apoptosis.

They also examined whether caspase inhibitors could rescue the cells from apoptosis induced by staurosporine. For this question they used the noncompetitive inhibitor of caspases z-VAD.fmk. The addition of z-VAD.fmk delayed the staurosporine-induced cell death [38].

The Bcl2 protein family plays a key role in the process of the intrinsic apoptotic signaling pathway, because dysfunctional Bcl2 protein may result in the development of both tumor cells and resistance to chemotherapies [39]. As a kinase inhibitor, staurosporine seems to be able to particularly influence the process of the intrinsic apoptotic signaling pathway.

In summary, this study showed that staurosporine induces apoptosis in pancreatic carcinoma cells via the intrinsic signaling pathway. Staurosporine is therefore a suitable positive control for in vitro apoptosis tests for the pancreatic carcinoma cell lines PaTu 8988t and Panc-1. In the colorectal cancer cell line SW 480, stimulation with staurosporine did not induce apoptosis.

Modern therapeutic strategies for tumor diseases target the efficient modulation of specific signaling and transcription pathways (for instance, VEGF antibodies [40], tyrosine kinase inhibitors in the treatment of chronic lymphatic leukemia [41], or EGFR antibodies in the therapeutic management of advanced colorectal carcinoma [42]). In this respect, the therapeutic potential of protein kinase inhibitors has been repeatedly discussed. Pharmaceutical companies increasingly focus on the development of new chemotherapies [43]. The approval of chemotherapies (for instance, imatinib for treating chronic myeloid leukemia, trastuzumab for treating breast cancer, or gefitinib and cetuximab for treating lung and colorectal cancer) has opened up new possibilities of treating different types of cancer [44]. A large number of kinase inhibitors are currently undergoing clinical development in both clinical and preclinical trials to analyze the potential of these substances for medical treatment [45]. However, their side effects and toxicity should be closely monitored because kinase inhibitors may also modulate important signaling cascades in healthy cells.

Ultimately, the basis and prerequisite of such new therapeutic approaches is a comprehensive knowledge of the respective carcinogenesis. The only types of cancer that may benefit from therapeutic protein kinase inhibitors are diseases marked by upregulation of the specific signaling pathways and thus by disturbed natural balance between mitosis and apoptosis. Furthermore, a combination therapy of different target-specific therapeutics will probably be required to avoid an increase in the proliferation rate or a decrease in the apoptosis rate of cells and thus the development of uncontrolled cellular growth [44].

The present work provides insight into the complexity of the Bcl2 family and the apoptotic pathways. Many further trials will be required to identify the underlying molecular mechanisms. Identifying and characterizing cellular receptors and their signal-transduction cascades will eventually help establish new therapeutic approaches in the treatment of pancreatic carcinoma, one of the most aggressive types of all cancers.