Differential Regulation of Cytochrome C Release in Dexamethasone-Resistant 7 TD 1 Cells

Interleukin-6 (IL6)-triggered JAK/STAT3 and PI3K/AKT signaling pathways are known to mediate cell survival, drug resistance and progression in a variety of cancer cells. Resistance to induction of apoptosis plays a critical role in the pathogenesis of numerous cancers and development of resistance to chemotherapeutic agents used in its treatment. Previous research in our laboratory employing a dexamethasone-resistant subline (7TD1-Dxm) of IL6-dependent 7TD1 cells indicated that constitutively activated STAT3 was important in control of apoptosis and targets downstream to activated STAT3 appeared to be involved in the development of resistance to dexamethasone by 7TD1 cells. We therefore investigated the hypothesis that Dxm-resistance developed by 7TD1-Dxm cells was due to resistance to induction of apoptosis mainly because of the dysregulation of the downstream targeted in JAK/STAT3 signaling pathway. Our results indicate that 7TD1-Dxm cells show resistance to Dxm-induced reduction of Bcl-2 protein and the release of cytochrome c. Thus, this study suggests that development of resistance to dexamethasone by 7TD1 cells may involve altered regulation of mitochondrial anti-apoptotic proteins.


Introduction
Interleukin-6 (IL6) is a pleiotropic cytokine involved in the progression of various cancers like colon, pancreatic and prostate cancers and multiple myeloma (MM) [1][2][3][4][5][6][7].It is known to mediate development of drug resistance in prostate cancer and MM [4][5][6][7].For instance, IL6 promotes cell proliferation and protects from dexamethasone (Dxm)-induced apoptosis by triggering three main signaling pathways including JAK/STAT3, PI3K/AKT, and MAPK signaling pathways [8][9][10][11].These factors contribute to the cells' development of resistance to a variety of chemotherapeutic agents.These observations are important to our understanding of the molecular mechanisms leading to drug resistance, especially to chemotherapeutic agents like dexamethasone and may lead us to find potential targets that can induce apoptosis in Dxm-resistant cells.
We have developed a dexamethasone-resistant subline of IL6-dependent 7TD1 cells (7TD1-Dxm) by chronic treatment with dexamethasone.Our previous findings suggested that STAT3 constitutive activation in 7TD1 cells was important for control of apoptosis but mechanisms for dexamethasone resistance were located downstream of STAT3 activation, and may include targets like Bcl-2, Bcl-X L and Mcl-1 [22].We therefore hypothesized that Bcl-2 family proteins may be contributing to development of resistance to dexamethasone by 7TD1 cells.Our strategy to investigate this hypothesis was to elucidate the effect of inhibition of Bcl-2 protein on inhibition of proliferation, induction of apoptosis, release of cytochrome c and its consequence on the mitochondrial membrane potential (MMP).Thus, this study aimed at

Cell Culture
7TD1 cells were grown in complete culture medium as described previously [22,23].The cells were cultured with 4 ng/ml recombinant human IL6 (Upstate Biotechnology, Lake Placid, NY).Chronically treated cells (7TD1-Dxm) were continuously cultured in the presence of dexamethasone (85 μM) and in the absence of IL6 over a period of three months to generate the resistant subline, 7TD1-Dxm.This subline was maintained in culture with dexamethasone (85 μM) and without IL6.Dexamethasone was dissolved in ethanol at 1 mg/ml stock solution.HA-14-1 (Sigma Aldrich, St.Louis, MO) was dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 50 mM.Appropriate vehicle controls were included in experiments.Cellular proliferation was monitored using the MTT assay [22,23] as described previously [24].All experimental procedures were carried out with a prior wash-out period of 24 hrs during which both the cell lines were incubated without IL6 or Dxm.All experiments utilized parent 7TD1 cells between passages 30 and 60.

Western Blotting
As previously described [22], cells were harvested by centrifugation at 200 × g for 6 minutes and whole cell lysates prepared using a lysis buffer containing 20 mM Tris-HCl (pH 7.6), 100 mM sodium chloride, 2% Triton X-100, 10 mM EDTA (Ethylene diamine tetraacetic acid), 100 mM sodium fluoride, 60 mM sodium pyrophosphate, 0.2% sodium azide, 0.2 mM sodium orthovanadate, 1 mM PMSF (Phenylmethanesulfonyl fluoride), 20 µg/ml leupeptin, and 2 µg/ml aprotonin.Lysates were subsequently sonicated for 5 seconds and centrifuged at 5200 × g for 10 minutes; the resulting supernatant was mixed with 3× SDS loading buffer and incubated at 90˚C for 3 minutes.The proteins were then separated on a 10% (w/v) polyacrylamide gel.Separated proteins were transferred to a PVDF membrane by electroblotting at 100 V for 1 hr.After transferring, the PVDF membrane was blocked in a solution containing 5% milk powder and 2.5% bovine serum albumin (BSA) in 1× Tris-buffered saline with 0.05% tween (TBST) at 4˚C for 6 hrs and then washed with 1× Tris-buffered saline (TBS) and 1× TBST for four and three times, respectively.After washing, the membrane was probed with primary antibody for total Bcl-2 protein and Bcl-X L (1:1000 dilutions, Cell Signaling Technology, Danvers, MA) at 4˚C overnight, followed by washing with 1× TBS and 1× TBST for four and three times, respectively.The membrane was then probed with Horse-Radish Peroxidase (HRP) labeled goat anti-rabbit IgG secondary antibody (1:2500 dilutions, KPL, Gaithersburg, MD).Blots were then processed by Pierce chemiluminiscence kit (Thermo Scientific, Rockford, IL) as recommended by the commercial vendor and autoradiographed on an X-ray film (RPI corp, Mt.Prospect, IL).Western blot analysis was carried out in triplicates.

Detection of Apoptosis
Apoptotic cells were quantified using the DNA fragmentation TUNEL assay (Molecular Probes, Eugene, OR).Briefly [22], 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, incubated for 48 hrs, washed with 1× PBS, fixed in a mixture of ice-cold 1× PBS: 70% ethanol, and kept at −20˚C for 12 -18 hrs.The cells were washed with 1× PBS, stained using the APOBrdu TUNEL assay kit according to the manufacturer's protocol, and analyzed using a flow cytometer (Becton Dickinson, San Jose, CA).

Cytochrome C Release Assay
Cytochrome c release into cytoplasm was quantified using a cytochrome c release assay kit (Calbiochem, La Jolla, CA).Briefly, 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, incubated for 24 hrs, washed twice with 1× PBS, resuspended in 300 µl permeabilization buffer and incubated on ice for 10 minutes.After permeabilization, the cells were fixed in 8% paraformaldehyde in PBS for a period of 20 minutes at room temperature.After fixing, the cells were washed twice in 1× wash buffer and resuspended in 250 µl of blocking buffer and kept at room temperature for 1 hr.After incubation, 250 µl of anti-cytochrome c antibody working solution was added to each sample and incubated for 1 hr at room temperature.The cells were then washed twice with 1× wash buffer.They were then resuspended in 500 µl of anti-IgG FITC and incubated again for 1 hr at room temperature.They were then washed with 1× washed buffer and resuspended in 400 µl of wash buffer and analyzed using flow cytometer (Becton Dickinson, San Jose, CA).

Determination of Mitochondrial Membrane Potential (MMP)
MMP was analyzed using two Mitotracker dyes Mitotracker red and Mitotracker green (Molecular probes, Eugene, OR).Mitotracker red binds to non-depolarized mitochondria, and Mitotracker green binds to all mitochondria.Thus, a ratio of non-depolarized to total mitochondria was calculated to estimate the MMP.Briefly, 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, and incubated for 24 hrs.At the end of indicated time point, Mitotracker red and Mitotracker green dyes were added for a final concentration of 250 nM and 150 nM, respectively.They were then incubated at 37 º C for 30 minutes, centrifuged at 200 × g for 6 minutes, washed and resuspended in 1× ice cold, PBS and analyzed by flow cytometer (Becton Dickinson, San Jose, CA).The ratio of mean red and green fluorescence intensities is expressed relative to the control group.

Statistical Analysis of Data
Statistical significance of experimental results was analyzed by univariate analysis of variance followed by Tukey's post-hoc test with a minimum significance level set at P < 0.05.In figures, individual letters identify those treatment groups where the differences are statistically significant as indicated by ANOVA (e.g., all treatment groups identified by a letter "a" are statistically significantly different from those treatments identified by all other letters, "b", "c", etc.).

Proliferation of Dxm-Resistant 7TD1 Cells Was Inhibited in a Concentration-Dependent Manner upon Treatment with HA-14-1, a Bcl-2 Specific Inhibitor
Consistent with our previous observations [22], 7TD1-Dxm cells were resistant to inhibition of proliferation by dexamethasone treatment as compared to parent 7TD1 cells.To examine the role of inhibition of Bcl-2, an antiapoptotic protein, on proliferation of 7TD1-Dxm cells, we initially treated 7TD1-Dxm cells with HA-14-1, a Bcl-2 specific inhibitor.Figure 1 shows that treatment with HA-14-1 inhibited proliferation of 7TD1-Dxm cells in concentration-dependent manner with maximum inhibition observed at 20 µM (a 4-fold decrease was observed).

Treatment with HA-14-1 at a Concentration That Showed Maximum Inhibition of Proliferation Induces Apoptosis in Dxm-Resistant 7TD1 Cells
We next examined the effect of HA-14-1 on induction of apoptosis.Figure 2 shows that treatment of both sensitive and resistant cells with 20 µM HA-14-1 alone had a significant impact on induction of apoptosis (40% -60% apoptotic cells were seen as compared to control) in both the lines.Addition of dexamethasone with HA-14-1 did not have further effect on induction of apoptosis as compared to treatment with HA-14-1 alone.This finding suggests that inhibition of Bcl-2 at the concentration required for maximum inhibition of prolif-  eration was sufficient to induce apoptosis but did not alter the resistance of 7TD1-Dxm cells to dexamethasone treatment.

7TD1-Dxm Cells Were Resistant to Reduction of Bcl-2 Protein Expression upon Treatment with Dexamethasone
Since inhibition of Bcl-2 by HA-14-1 blocked proliferation and induced apoptosis in both parent 7TD1 and 7TD1-Dxm cells, we next examined Bcl-X L and Bcl-2 protein expression.cells were completely resistant to reduction of Bcl-2 protein upon treatment with Dxm.This effect of reducing Bcl-2 appears to be specific as Bcl-X L was unaffected by Dxm treatment (Figure 3(b)).This observation led us to propose a role for dexamethasone in differential regulation of the anti-apoptotic proteins downstream to JAK/ STAT3 signaling pathway in the process of acquisition of the Dxm-resistant phenotype and confirmed our assumption about the involvement of mitochondrial pathway of apoptosis induction in the development of resistance in these cells.Parent 7TD1 cells were treated with IL6 at 4 ng/ml which was used as a negative control.The densitometric analysis of the results obtained from Bcl-2 western blot analysis (Figure 3(c)) shows that while in the parent 7TD1 cells the protein levels of Bcl-2 significantly decreased upon treatment with dexamethasone alone, in 7TD1-Dxm cells Bcl-2 protein levels were resistant to reduction by dexamethasone.

Dexamethasone-Resistant 7TD1 Cells Were Resistant to Cytochrome C Release upon Treatment with Dexamethasone
We next examined the effect of dexamethasone on cytochrome c release from mitochondria into the cytoplasm which is a downstream event that occurs after reduction of Bcl-2 protein.Histogram analysis of the data obtained from flow cytometric analysis suggested while there was a shift in the peak observed in parent 7TD1 cells treated with dexamethasone alone at 85 µM when compared to control, suggesting a release in the cytochrome c into the cytoplasm, no shift was observed in 7TD1-Dxm cells treated with dexamethasone alone as compared to control (Figures 4(a) and (b)).Figures 4(c) shows that treatment with HA-14-1 at 20 µM alone and in combination with 85 µM dexamethasone induced significant release of cytochrome c into the cytoplasm (up to 70% labeled cells were observed) indicating the role of Bcl-2 in regulating the release of cytochrome c from the inter-membranal space of mitochondria.Upon treatment with dexamethasone alone, 7TD1-Dxm cells showed resistance to release of cytochrome c while the parent 7TD1 cells showed a significant labeling of the cells (40% labeled cells observed compared to control) suggesting release of cytochrome c into the cytoplasm.This result is consistent with the effect of Dxm treatment on Bcl-2 protein expression and suggests a role for mitochondria in the development of resistance by these cells.

Mitochondrial Membrane Potential Disruption Was Not Observed in Both Parent 7TD1 and Dxm-Resistant 7TD1 Cells
Since cytochrome c release was not observed in 7TD1-Dxm cells upon dexamethasone treatment, we further determined the MMP as its disruption can be a prior step for release of cytochrome c.We treated both cell lines with IL6 (4 ng/ml), AG490 at 50 µM, HA-14-1 at 20 µM, alone and in combination with dexamethasone (85 µM) or with dexamethasone alone at 85 µM for a period of 24 hrs. Figure 5 shows that both parent 7TD1 cells and 7TD1-Dxm cells were resistant to inhibition of MMP suggesting that MMP disruption was not responsible for releasing cytochrome c.Thus, other mechanism(s) are likely involved.Similar results were obtained even when the incubation period was extended to 48 or 72 hrs.

Discussion
Several studies have shown that changes in expression of Bcl-2 family proteins may lead to chemotherapeutic resistance and vigorous growth of myeloma cells in the bone marrow microenvironment (BMM) [12,14,[16][17][18].Our previous studies have indicated a role of constitutively activated STAT3 in controlling induction of apoptosis in 7TD1-Dxm cells [22].In the current study, we focused on investigating the role of downstream targets of activated STAT3 in the development of resistance to dexamethasone in 7TD1-Dxm cells.We found that treatment with HA-14-1 alone, a Bcl-2 specific inhibitor significantly inhibited proliferation of 7TD1-Dxm cells at 20 µM itself and also induced apoptosis in both parent 7TD1 and 7TD1-Dxm cells (Figures 1 and 2).The sensitivity of the 7TD1-Dxm cells was not altered when treated in combination with dexamethasone.Over-expression of the anti-apoptotic proteins such as Bcl-2 has been shown to play a role in the survival of MM cells and development of drug resistance [12,15].Gauthier et al. have investigated the role of Bcl-X L in the control of apoptosis in murine myeloma cells [25].Quintinella-Martinez et al. have noted the relationship between IL6, STAT3 activation and Bcl-2, Bcl-X L expression and their role in promoting proliferation of myeloma cells [21].
Therefore, we focused our current investigations on the regulation of Bcl-2 protein expression.Our results showed that 7TD1-Dxm cells were resistant to reduction of Bcl-2 protein upon treatment with dexamethasone alone, unlike the parent 7TD1 cells which showed significant reduction of protein levels of Bcl-2 when treated with Dxm (Figure 3  cocorticoids), MM.1R E (resistant to treatment with glucocorticoids), MM.1R L (resistant to treatment with glucocorticoids) in order to study the progression of MM and the mechanisms responsible for resistance to glucocorticoids including dexamethasone [40].A study conducted by Rosen et al., using the MM cell lines showed that apoptosis induction by glucocorticoids like dexamethasone was mediated through glucocorticoid-induced leucine zipper (GILZ) [41].Furthermore, Rosen et al. found that PI3K/AKT signaling pathway played a role in the regulation of GILZ and indicated that the use of glucocorticoid in combination with PI3K inhibitor may be useful in treating clinical resistance of multiple myeloma [41].Our present findings demonstrate that resistance to dexamethasone in 7TD1 cells is accompanied by the inhibition of cytochrome c release from the mitochondria upon treatment with Dxm.There may be, however, other mechanisms responsible for the development of Dxmresistance.Nevertheless, our finding is novel in that we have established a Dxm-resistant phenotype of 7TD1 cells in the absence of IL6 and they are also IL6-independent for growth.Employing this phenotype we have elucidated a potential mechanism responsible for development of resistance to dexamethasone.Clearly, this is an important area that deserves further investigation. of mitochondria.Disruption of mitochondrial membrane potential and release of cytochrome c from the mitochondrial inter-membranal space are pre-requisite steps to induce the sequential events leading to cell death.Our results indicated that 7TD1-Dxm cells were resistant to release of cytochrome c from the mitochondria while the parent 7TD1 cells showed a significant increase in the percent labeled cells indicating the release of cytochrome c upon treatment with dexamethasone (Figure 4).
Since cytochrome c release is known to be accompanied with a change in the mitochondrial membrane permeability [12,26,27], we next investigated the regulation of MMP in these cells.We found that MMP was not disrupted during treatment with dexamethasone, in either parent 7TD1 or 7TD1-Dxm cells (Figures 5).This finding indicates that there may be ways for cytochrome c release other than the disruption of MMP leading to formation of membrane transition pores (MPT).Indeed, Von Ahsen et al. have suggested there were ways other than disruption of MMP for the release of cytochrome c [27].A number of studies have also suggested that disruption of the MMP was not required for cytochrome c release [28][29][30][31][32].Thus our results are consistent with the possibility that mitochondria can be induced to release their cytochrome c without disruption of their membrane potential.

Figure 2 .
Figure 2. Treatment of 7TD1-Dxm cells with HA-14-1 induced apoptosis but did not alter resistance to dexamethasone.7TD1-Dxm cells were treated for 48 hrs either with 20 µM HA-14-1, alone or with 85 µM dexamethasone in the presence of 20 µM HA-14-1.After 48 hrs treatment, cells were evaluated for apoptosis using the TUNEL assay and DNA fragmentation therein analyzed by flow cytometry.Values represent the average percent apoptotic cells ± S.D. for triplicate assays.Etoposide, at 10 µM, was used as a positive control.

Figure 3 (Figure 3 .
Figure 3. (a) Dexamethasone-resistant 7TD1 cells were resistant to reduction of protein levels of Bcl-2.7TD1 cells were treated with/without interleukin-6 (4 ng/ml) and 7TD1-Dxm cells without IL6 and both the cell lines treated with 50 μM AG490 or 85 µM dexamethasone and incubated for 48 hrs at 37˚C.After incubation, cells were collected and lysates prepared and subjected to Western blot analysis for detection of Bcl-2 protein levels.The experiment was repeated three times; (b) Protein levels of Bcl-X L were unaffected by dexamethasone treatment in 7TD1 cells.7TD1 cells were treated with/without interleukin-6 (4 ng/ml) and 7TD1-Dxm cells without IL6 and both the cell lines treated with 50 μM AG490 and 85 µM dexamethasone and incubated for 48 hrs at 37˚C.After incubation, cells were collected and lysates prepared and subjected to Western blot analysis for detection of Bcl-X L protein levels.The experiment was repeated three times; (c) Densitometry analysis of results from Western Blot analysis of Bcl-2 protein.Average pixel intensity of each band was obtained by using Un-ScanIt software.The values were then normalized with βactin, positive control and percent control calculated for each value.Values represent mean ± S.D. of duplicate values from triplicate samples of pixel intensities as obtained from densitometric analysis and normalized to actin.

Figure 4 .
Figure 4. Histogram analysis of flow cytometric data for release of cytochrome c into the cytoplasm.(a) Parent 7TD1 cells where Purple peak represents control, Red represents HA-14-1 treatment alone at 20 µM concentration, Yellow represents treatment with HA-14-1 at 20 µM in the presence of dexamethasone at 85 µM concentration, and Blue represents treatment with dexamethasone alone at 85 µM concentration; (b) 7TD1-Dxm cells where Purple peak represents control, Red represents HA-14-1 treatment alone at 20 µM concentration, Green represents treatment with HA-14-1 at 20 µM in the presence of dexamethasone at 85 µM concentration, and Yellow represents treatment with dexamethasone alone at 85 µM concentration; (c) Cytochrome c release from the mitochondria was not observed in the 7TD1-Dxm cells upon treatment with dexamethasone.Both 7TD1 and 7TD1-Dxm cells were treated with either dexamethasone (85 µM) alone or HA-14-1 at 20 µM alone or HA-14-1 in combination with dexamethasone and incubated for 24 hrs at 37˚C.After 24 hrs the cells were subjected to a cytochrome c release assay following the manufacturer's protocol and analyzed by flow cytometer (Becton Dickinson, San Jose, CA).Values represent the average percent apoptotic cells ±S.D. for triplicate assays.
(a)).No observable changes were detected in Bcl-X L protein levels in both cell lines (Figure 3(b)).Detection of Mcl-1 (Myeloid Cell Leukemia factor-1), an anti-apoptotic protein known to play a role in the survival of MM cells [15], suggested that it was constitutively expressed in both parent 7TD1 and 7TD1-Dxm cells, both at the transcriptional-and translationallevel (data not shown).When combined, our results suggested a further investigation of the role of mitochondria in development of resistance to dexamethasone by 7TD1-Dxm cells.It is known that Bcl-2 protein controls apoptosis induction and/or inhibition by regulation at the level

Figure 5 .
Figure 5. MMP disruption was not observed in both parent 7TD1 and 7TD1-Dxm cells.Both 7TD1 and 7TD1-Dxm cells were treated with IL6 at 4 ng/ml or AG490 at 50 µM or HA-14-1 alone at 20 µM and in combination with dexamethasone or with dexamethasone alone in a time-dependent manner.Cells were analyzed using flow cytometer after 24 hrs.After each timepoint cells were collected, Mitotracker red and Mitotracker green dyes were added for a final concentration of 250 nM and 150 nM, respectively.They were incubated at 37˚C for 30 min and washed twice with ice cold 1× PBS at 200 × g for 6 min., and resuspended in 400 µl of 1× PBS and analyzed by flow cytometer (Becton Dickinson, San Jose, CA).Values represent the ratio of average of mean fluorescence values from non-depolarized to total mitochondria, ±S.D. for triplicate assays, normalized to control.
. Previously, Rosen et al. have established three different MM cell lines; MM.1S (sensitive to treatment with glu-This study was supported by NIH Grant P20RR16454 from the INBRE program of the National Center for Research Resources.