Mechanisms Of Lidamycin Towards Human Colorectal Cancer Cells With Different P53 Gene Status

Enediyne antibiotics have been reported to be the most potent cytotoxic anti-tumor agents. Lidamycin (LDM, originally named C-1027) is a member of the enediyne antibiotics family. LDM consists of a chromophore and an apoprotein, and the former has high ability to attack DNA, whereas the latter plays the role as a protecting protein. P53 is well known as a tumor suppressor and plays an important role in inducing cell apoptosis and cell arrest. But the role of p53 in the pathway by which LDM induce apoptosis of tumor cells remains to be elucidated. In this report, we studied the complicated mechanisms of apoptosis induced by LDM and displayed both p53-dependent and -independent apoptosis initiated by this compound in human colorectal cancer cells in a dose-specific manner.1. LDM induces apoptosis in a panel of human colon cancer cell lines with different p53 statusColon cancers are one of most popular cancers in human with high mortality and genetic complexity. P53 is frequently mutated in approximately 60% of colon cancers⁽¹⁾. To evaluate the effect of p53 status on the response to LDM, we treated a panel of human colon cancer cell lines harboring wild type or mutated p53 with LDM. Cell cytotoxcity or death was determined following 24h treatment at various doses from 1 nM to 1μM by MTT or flow cytometric analysis (FACS) respectively. Accumulation of viable cells as measured by MTT was used to assess the cytotoxicity of LDM, while FACS was employed to determine the rate of sub-G1 rate, which is representative of apoptosis. As shown through MTT redults, in general, tumor cells that carry wild type p53 (HCT116, LOVO, COLO205) were more sensitive to LDM induced toxicity at 10 nM level (growth inhibition range: 50-70%) when compared with those carrying p53 mutations (HCT15, SW620, SW480, HT29) (10-25%). However, this p53 dependence was lost when LDM reached up to 1μM. LDM induced toxicity can be resulted from either cell death or proliferation arrest. The parallel trends were observed by FACS analysis, indicating that LDM exerts its cytotoxic effects primarily through induction of apoptosis.2. LDM induces both p53-dependent and p53-independent apoptosis in HCT116 cellsTo further determine the role of p53 in response to LDM, we studied the effects of LDM on a pair of isogenic colon cell lines, HCT116 p53 wild type (wt) and p53 knocked out (ko), with the latter derived form parental HCT116 by targeted homologous recombination⁽²⁾. HCT116 colon carcinoma cells are mismatch defective, poor differentiated and growth-factor insensitive yet responsive to a variety of stresses⁽³⁾. A key mediator of stress response is p53 in wild type HCT116 cells that is stabilized and activated upon various stress stimulation. HCT116 p53 wt and ko cells were treated with LDM at various doses for 24h before subjected to FACS or MTT analysis. The results clearly demonstrated a primarily p53-dependent apoptosis is induced significantly after 24h exposure to LDM of 10 nM assayed in either method, and this p53 dependence is time-reliant within 24h. When the dose of LDM was increased to 100 nM or even higher, p53 ko cells become as sensitive to LDM induced cell death as p53 wt cells. Actually, the loss of p53 dependence occurs as early as within 6h after treatment with LDM of higher concentration. This p53 dependence is further manifested by the activation of p53 protein and function indicated by p21 up-regulation in a time-dependent manner in HCT116 p53 wt cells after exposure to LDM at relatively low but not high concentration, indicating the interestingly primary p53 dependence in a dose-specific mechanism. Notably, there exist around 15% of apoptotic cells in HCT116 p53 ko cells after treated with LDM of 10 nM, suggesting a trivial p53 independent mechanism occurs in low dose LDM induced apoptosis.Next, we measured the IC50 of LDM by MTT assay in both cell lines, and compared the IC50 of LDM with other frequently used chemotherapeutic drugs such as 5-fluorouracil (5-Fu), mitomycin C (MMC) and adriamycin etc. IC50 of LDM is at least three orders of magnitude lower than that of other agents, indicating its much higher activity towards tumor cells. The IC50 of LDM in HCT116 p53 wt cells is much lower than that in ko cells, demonstrating the relative resistance to LDM induced toxicity in the absence of p53. The similar p53-dependent sensitivity towards cell death was observed in response to 5-fluorouracil (5-Fu) and mitomycin C (MMC) while the toxicity of taxol towards HCT116 cells seems to have no association with p53 status. Furthermore, p53 takes more protective effects on Adriamycin induced cell death when compared with loss of function cells.As stated in many studies, one characteristic of apoptosis is DNA fragmentation and chromatin condensation. We observed 10 nM LDM mainly induced typical chromatin condensation manifested by cellular shrinkage and apoptotic body formation, which is mainly reliant on p53-dependent signaling pathway (with the similar apoptotic features induced by MMC), while the higher concentration of LDM induced atypical "dotted" chromatin condensation, which is p53-independent and correlates more closely with DNA cleavage activity of LDM. These data were further supported by TUNEL staining that demonstrated DNA fragmentation with similar trends. Results of DNA ladder were similar to chromatin condensation and TUNEL staining. 10 nM LDM could incise DNA at 4h in p53 wt cells which was early than p53 ko cells (12h). But 1μM LDM could induce DNA ladder as early as 15min and had no difference in two cells. Taken together, we conclude that LDM of low dose mainly initiate apoptosis through p53-dependent signaling pathway, whereas LDM of high dose takes effect in a more direct and swift way regardless of p53 status.3. LDM-induced apoptosis is mediated through mitochondria activated pathwayMitochondria are central death regulators in response to DNA damage⁽⁴⁾ and are especially critical for p53-dependent apoptosis^(5-7). To determine the involvement of mitochondria pathway, we measured the loss of mitochondria transmembrane potential (△Ψm), which is often a general indicator in mitochondria activation⁽⁸⁾. As indicated in results, 12 hours exposure to 10 nM LDM resulted in a gradually drop of△Ψm in a primarily p53-dependent manner as determined by the fluorescence dye dihydrhodamine 123. The loss of△Ψm clearly precedes DNA fragmentation that reached only about 18% at this time. LDM of higher dose didn't influence the△Ψm within 6h. The data suggests that LDM induces activation of mitochondria formed signaling pathway in a strict concentration-dependent manner. The involvement of mitochondria pathway is further supported by the generation of reactive oxygen species (ROS), which usually act upstream of disruption of△Ψm in p53-induced apoptosis⁽⁹⁾. A sharp dose-responsive increase of ROS was observed in HCT116 p53 proficient cells rather than in p53-deficient cells. The activation of mitochondria pathway was substantiated by western analysis. We observed the activation of caspase-9, in p53 wt cells after treated with LDM at 10 nM, which plays a key role in the initiation of mitochondria apoptotic pathway by forming apoptosome complex with cytochrome c and Apaf-1⁽⁴⁾. Caspase-3 6 and 7 was thereafter activated and sequentially cleaved its downstream target PARP from 116kDa into 89 kDa fragment, which is a well-established proapoptotic event.It should be pointed out that p53-independent caspase-9, 3, 6 and 7 and PARP cleavage were also observed in p53 ko cells, although to a much less extent. This might account for the minor apoptotic phenotype induced by 10 nM LDM in p53 ko cells as described above.Obviously, caspase cascade wasn't activated in both cell lines after exposure to LDM of as high as 1μM, indicating there exists special mechanism other than typical mitochondria pathway at the suggested dose level.4. The role of Bcl-2 family members in LDM induced apoptosisExpressions of the Bcl-2 family proteins were analyzed such as PUMA, Bax, p-Bad Bax, Bak, Bim and Bcl-2. Increased expressions of PUMA, p-Bad, Bax, Bak and Bim were demonstrated in 10 nM LDM induced apoptosis through tightly p53-dependent and time-dependent way. However, no significant alteration of Bcl-2 expression was found under the same condition, which is reported to be another p53 transcriptional repressed target. The results suggest that the transcriptional activation function of p53 might play a major role in LDM induced apoptotic pathway.We further analyzed the role of proapoptotic proteins Bax and PUMA in LDM induced cell death by the use of HCT116 Bax-/- and PUMA-/- cell lines. As predicted, the sensitivity to LDM of 10 nM decreased dramatically in either Bax or PUMA-deficient cell lines when compared with wild type cells, indicating that Bax or PUMA indeed plays an important role in LDM induced apoptosis.Next, to further clarify the possible protective role of Bcl-2, we analyzed the effect of exogenous expression of Bcl-2 on the sensitivity of HCT116 p53-proficient cells towards LDM. As shown in results, exogenous expression of Bcl-2 decreased, but not totally blocked the sensitivity in HCT116 p53 wt cells to 10 nM LDM induced cytotoxicity. Our results indicated that Bcl-2 is only a partial but not a solely regulator of apoptosis induced by LDM under strict circumstances. To further establish the direct role of p53 in the regulation of LDM induced apoptosis, we transiently transfected p53 wild type plasmid into p53 ko cells to restore its p53 activity, and increased sensitivity of transfected cells was observed in response to LDM as of 10 nM level, though to a less extent than that in p53 wild type cells. This reflects that transient expression can only restore partially the activity of p53. As predicted, either Bcl-2 or p53 overexpression didn't change the phenotypes of HCT116 cells in response to LDM of as high as 1μM.5. LDM-indueed apoptosis is inhibited by caspase inhibitor in a dose-specific wayThe final executioners of apoptosis are activated by caspases in the downstream of mitochondria apoptotic signaling pathway, such as caspase-3, 6 and 7⁽¹⁰⁾. Our previous study has reported that LDM induced apoptosis in a caspase-independent way, although the caspases were activated at a relative late time⁽¹¹⁾. However, that experiment was performed in the range of 1μM. Here we demonstrated that LDM of low dose does induce apoptosis in a caspase-dependent way, because apoptosis induced by low dose LDM was totally blocked both in p53 wt and ko cell lines, indicating that LDM of low dose initiates apoptosis through an entirely caspase-dependent way. The caspases dependency of low dose LDM induced apoptosis occurs in a time-dependent manner within 24h. In consistence with our previous study, LDM of up to 1μM induces apoptosis in a caspase-independent way within 24 hours.The caspases dependence was further supported by western blot analysis which showed that pan-caspases inhibitor VAD-fmk completely blocked caspase-3 and caspase-9 activation as well as PARP cleavage in both HCT116 cell lines. Interestingly, the activation of p53 and Bax was reduced as well by VAD-fmk, reflecting a more complex feedback regulations might occur at the mitochondria pathway. DNA ladder results also showed that VAD-fmk could not inhibit the DNA rupture resulted from high dose LDM treatment.6. Low dose LDM induced apoptosis is mediated by altered expressions of kinases like p-MAPK and NF-κBMany studies have suggested the close correlation between p53 and kinases pathway such as MAPK and NF-κB. These protein kinase members participate in integrating extracellular signals to regulate cell proliferation, differentiation, cell survival and apoptosis^(12,13). Their possible roles in LDM induced apoptotic pathway have not been defined so far. To assess their possible involved functions towards LDM induced toxicity, we analyzed the expressions of activated MAPK and p65 subunit of NF-κB. Results indicated that low dose LDM significantly increases the activation of p-MAPK in a p53-dependent fashion while high dose doesn't. Results of other MAPK family showed that expressions of activated p-38 and p-JNK remain unchanged following similar treatment. Under the similar context, expression of p65 NF-κB was down-regulated obviously in both time- and dose-dependent manner. The decreased p65 subunit reflects the repressed transcriptional activity of NF-κB, which stimulates cell survival and promotes cell proliferation.7. Lidamycin induced G2/M arrest in HCT116 cells in a p53 independent mannerIn the meantime, it was suggested that LDM of as low as 0.1-1 nM mainly induces a significant and similar G2/M arrest in both p53 wt and ko cells. Western blot results showed that LDM induced G2/M arrest was associated with increasing expression of p53, p-cdc2 and increasing cyclin B1 expression in HCT116 p53 wt and ko cells.