Disrupted expression of CXCL5 in colorectal cancer is associated with rapid tumor formation in rats and poor prognosis in patients.

PURPOSE: We isolated a subline (CC531M) from the CC531S rat colon carcinoma cell line, which grows and metastasizes much more rapidly than CC531S. We found, using RNA expression profiling, that one of the major changes in the CC531M cell line was a 5.8-fold reduction of the chemokine CXCL5. The purpose of this study was to determine the effect of CXCL5 expression on colorectal tumor growth and metastasis. EXPERIMENTAL DESIGN: CC531 clones were generated with either knockdown or restored expression of CXCL5. These clones were inoculated in the liver of rats. In addition, in two independent cohorts of colorectal cancer patients, the level of CXCL5 expression was determined and associated to clinical variables. RESULTS: Knockdown of CXCL5 expression in CC531S resulted in rapid tumor growth and increased number of metastasis, whereas restored expression of CXCL5 in CC531M resulted in a return of the "mild" tumor growth pattern of the parental cell line CC531S. In vitro, no difference was found in proliferation rate between clones with either high or low expression of CXCL5, suggesting that environmental interactions directed by CXCL5 determine tumor outgrowth. Finally, the importance of our findings was established for patients with colorectal cancer. We found that low expression of CXCL5 was significantly associated with poor prognosis for colorectal cancer patients. CXCL5 showed a trend (P = 0.05) for a positive correlation with intratumoral CD8(+) T-cell infiltration, suggesting a possible explanation for the observed poorer prognosis. CONCLUSIONS: Our results show that CXCL5 is important in growth and development of colorectal cancer, implicating a future role in both cancer therapy and diagnosis.

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line.

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells.

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.

Regional loss of the mitochondrial membrane potential in the hepatocyte is rapidly followed by externalization of phosphatidylserines at that specific site during apoptosis.

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to approximately 30% and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.

Rat colon carcinoma cells that survived systemic immune surveillance are less sensitive to NK-cell mediated apoptosis.

In order to form distant metastases, cells from the primary tumor have to detach, enter the blood- or lymph-compartment and escape immune surveillance. Here, we describe the selection of rat colon carcinoma cell lines (CC531s-m1 and CC531s-m2) that escaped from systemic immune surveillance; CC531s cells were injected into the v. jugularis of Wag/Rij rats, after three weeks the lung tumors were isolated, the tumor cells were cultured, characterized and injected again. The m1- and m2-cell lines were less susceptible for killing by syngeneic NK cells. Further characterization of this cell line showed a decreased sensitivity towards TRAIL- and CD95L-, but not to granzyme B-mediated apoptosis. In the m1- and m2-cells log-phase growth started earlier as compared to the parental cell line, whereas no changes were found in anchorage-dependent or anchorage-independent growth. After subcapsular injection of the m2-cell line into the liver of rats much more lung metastases were formed in comparison to injection of the parental cell line. In conclusion, the results suggest that the resistance of the m1- and m2-cells to NK cell-mediated apoptosis was associated with their capability to survive systemic immune surveillance and form metastases in vivo.

Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.

The role of calpains in apoptotic changes in isolated hepatocytes after attack by Natural Killer cells.

Previously, we showed that interleukin-2 activated Natural Killer cells (A-NK cells) in vitro rapidly induced apoptosis in freshly isolated rat hepatocytes (Blom et al., 1999. Hepatology 29 (3): 785-792) which was caused by a rapid decrease in the mitochondrial membrane potential and activation of caspases. In the present study we investigated the involvement of calpains in A-NK cell-induced apoptosis in isolated hepatocytes. When NK cells and hepatocytes were incubated in the presence of a calpain inhibitor the number of apoptotic cells decreased from 46 to 36%. However, more hepatocytes became necrotic (48 vs. 30%) as compared to the uninhibited situation. Inhibition of the calpains alone could not prevent the induction of the nuclear and cytoskeletal disruptions occurring in the hepatocytes. Inhibition of both calpains and caspases increased the number of necrotic cells as compared to incubation with a single inhibitor. However, the damage to the cytoskeleton of the surviving cells was completely inhibited. We conclude that calpains play a role in induction of apoptosis by NK cells. However, their role is limited as compared to caspases.