In response to Cisplatin no apoptosis is detectable in MSP and MSP RAS cells 54

To address the question, whether EMT affects the response to chemotherapy HMLE and HMLE RAS cells were exposed to Cisplatin for 8h or 16h. Thereupon, cells were harvested and apoptotic markers were identified by western blot. Activation of Caspase 3 (cleaved Caspase 3) and cleavage of PARP imply apoptosis induction after chemotherapeutic treat-ment. These apoptotic mediators were detectable for 24⁺HMLE as well as for 24⁺ RAS cells, but not for MSP or MSP RAS cells (Figure 5.2B, C). Even higher concentrations of Cisplatin (60µM) did not result in generic detached rounded cells in case of MSP cells (Figure 5.2A).

As an alternative measure to define apoptotic cells, activated caspases were determined by flow cytometry. MSP RAS cells treated with 30µM Cisplatin displayed less amounts of apop-totic cells compared to 24⁺ RAS cells (Figure 5.2D), indicating lack of activation of apoptotic mediators in MSP and MSP RAS cells after Cisplatin treatment.

Figure 5.2: In response to Cisplatin no induction of apoptosis is detectable in MSP RAS cells.

(A) 16h after seeding HMLE cells were treated with 40µM or 60µM Cisplatin for 8h. Thereafter, morphology of cells was checked with phase-contrast microscopy. (B, C) HMLE (B) and HMLE RAS cells (C) were treated with 20µM or 40µM Cisplatin for 16h. Cells were harvested and lysates were analyzed by western blot. Actin

staining was used as loading control and arrows are indicating the intended staining. One representative experi-ment of n=3 is shown. (D) 16h after seeding HMLE RAS cells were treated with 30µM Cisplatin for 16h. Cells and supernatants were harvested and apoptotic cells were determined using the Guava® Multi Caspase FAM Kit and flow cytometry. Data show mean ±SD of n=3.

It was recently published that EMT activators are able to directly manipulate the cell-cycle progression impairing proliferative activity [122; 123]. Diminished cell proliferation is as-sumed to protect from DNA damage and would result in delayed or missing induction of apoptosis upon chemotherapeutic treatment [124]. We therefore performed cell proliferation assays in HMLE as well as HMLE RAS cells. Proliferation was measured over 2d by direct cell counting, defining the cell number per well using a Celigo® cytometer. The analysis showed that 24⁺HMLE cells displayed different growth rates compared to MSP cells, while they were comparable for HMLE RAS cells (Figure 5.3A, B mock). Implicating that different growth rates are not the reason for lacking apoptosis, we decided to use the HMLE RAS cell system for further analysis. To this end, HMLE RAS cells were treated with 5µM or 10µM Cisplatin for 24h and subsequently, Cisplatin was replaced by fresh medium and the cell pro-liferation were measured over 7d every 24h by direct cell counting. Strikingly, MSP RAS cells continued their proliferation 5d after Cisplatin withdrawal, while 24⁺ RAS cells did not recover (Figure 5.3B). Considering these results, we were interested to know whether Cispla-tin is absorbed by MSP RAS and explored the CisplaCispla-tin adducts that were formed between the platinum atom and DNA after Cisplatin treatment. We made use of an Immuno-Slot-Blot as-say in cooperation with J. Thomale, University of Duisburg-Essen, applying an antibody that specifically recognizes the platinum atom bound to GpG [14]. These data demonstrated that 24⁺ RAS and MSP RAS display equal amounts of Cisplatin adducts after treatment (Figure 5.4). This experiment argues that DNA of 24⁺ RAS cells and MSP RAS cells is damaged to the same degree by chemotherapy. Thus, neither different cell proliferation nor Cisplatin transport processes are involved in apoptosis resistance of MSP RAS cells.

Figure 5.3: MSP RAS cells recover from Cisplatin treatment.

(A) 8h after seeding HMLE cells, direct cell counting was performed for 48h by defining the cell number per well using a Celigo® cytometer. One representative experiment of n=3 is shown. (B) 24h after seeding HMLE RAS were treated with 5µM or 10µM Cisplatin for 24h. Afterwards, cells were washed with supplemented MEGM medium and kept in culture. Cell number was measured once a day for 7d with a Celigo® cytometer af-ter adding fresh medium. Non-treated cells were used as control. Data show mean ±SD. One representative ex-periment of n=3 is shown.

Figure 5.4: Cells before and upon EMT display equal amounts of Cisplatin adducts.

24h after seeding HMLE RAS cells were treated with 20µM or 40µM Cisplatin for 16h. Afterwards, DNA was extracted with NucleoBond® AXG columns and analyzed for formed adducts by Immuno-Slot-Blot assay. Ex-periments were conducted with J. Thomale, University of Duisburg-Essen. Non-treated cells were used as con-trol. Data show mean ±SD of n=3.

5.1.3 MSP RAS cells display a general apoptosis deficit

Given the above results, we made use of different inhibitors manipulating distinct pathways that are involved in EMT or cell survival to test whether this would sensitize MSP RAS cells to Cisplatin treatment: First, we inhibited the Akt-signalling pathway by Akt and mTor inhibi-tors. Akt is a pro-survival protein, which is described as mediator of chemoresistance to Cis-platin [125]. Furthermore, we manipulated the mitogen activated protein kinase Mek and in-hibited JNK and p38, which are involved in the activated Wnt-signalling pathway [126]. MSP RAS cells were treated with the respective inhibitors in presence or absence of Cisplatin; sub-sequently, cells were harvested and analyzed for induced apoptosis mediators by western blot.

None of the treatments resulted in PARP cleavage; implicating no effects on the response to Cisplatin in MSP RAS cells (Figure 5.5A, C, D). Even by inhibiting TGFβ-induced pathways no changes concerning cleaved PARP were detectable for MSP RAS cells (Figure 5.5B). On that account, it was tested whether other chemotherapeutics were able to induce apoptosis in MSP RAS cells. To this end, HMLE RAS cells were exposed to Carboplatin, Doxorubicin or Neocarzinostatin (NCS) followed by western blot analyses for PARP cleavage. Strikingly, none of the chemotherapeutics induced apoptosis in MSP RAS cells while 24⁺ RAS cells showed PARP cleavage for every treatment (Figure 5.6). These findings raised the question whether it is possible to induce apoptosis in MSP RAS cells independent from DNA damage.

To test this, HMLE RAS cells were exposed to several concentrations of the death ligands Trail or TNFα. Since these ligands induce the transcription of NFκB and thus pro-survival pathways as a negative feedback, all treatments were performed in the presence of Cyclo-heximide a protein synthesis inhibitor preventing NFκB translation [127]. For 24⁺ RAS cells activation of Caspase 3 as well as PARP cleavage was detectable for both treatments. But even for higher concentrations of Trail no PARP cleavage was identified for MSP RAS cells (Figure 5.7A). Only for TNFα concentrations four times higher compared to the concentra-tions used for 24⁺ RAS cells, activation of the apoptosis mediators was observed (Figure 5.7B).

In summary, these results suggest that EMT results in MSP RAS cells that are resistant to the induction of apoptosis through chemotherapy or independent from DNA damage through death ligands.

Figure 5.5: Manipulation of distinct signalling pathways does not sensitize MSP RAS cells towards Cispla-tin.

(A-D) 24h after seeding HMLE RAS cells (A) or MSP RAS cells alone (B-D) were treated with the indicated inhibitors of different signalling pathways in the presence or absence of 20µM or 40µM Cisplatin for 16h. Af-terwards, cells were harvested and lysates were analyzed by western blot. Actin staining was used as loading control. Controls were treated with similar amounts of DMSO, n=1.

Figure 5.6: MSP RAS are resistant to a variety of chemotherapeutics.

(A-C) 16h after seeding HMLE RAS cells were treated with the indicated concentrations of Carboplatin for 16h (A), Doxorubicin for 24h (B) or NCS for 20h (C). Thereafter, cells were harvested and lysates were analyzed by western blot. Actin staining was used as loading control. Controls were treated with similar amounts of the re-spective solvent, n=1.

Figure 5.7: In response to the treatment with death ligands no apoptosis is detectable in MSP RAS cells.

(A, B) 16h after seeding HMLE RAS cells were treated with the indicated concentrations of Trail (A) or TNFα (B) in presence of 20µg/ml CHX for 6h. Subsequently, cells were harvested and lysates were analyzed by west-ern blot. Controls were treated with Ethanol for CHX or sterile H₂O for Trail and TNFα. Actin staining was used as loading control and arrows are indicating the intended staining. Shown is one representative experiment of n=3.