Introduction

Inflammation plays an important role in tumor initiation and progression1. Signal transducer and activator of transcription 3 (Stat3) is one of seven Stat proteins and can be activated by growth factors, cytokines, and oncogenic kinases in the inflammatory microenvironment including ultraviolet radiation, carcinogenic chemicals, stress and smoking2,3,4,5,6,7. Stat proteins, in particular Stat3, are highly activated in a number of cancer cell lines and human tumor samples8. It has been shown that constitutively active Stat3, but not a dominant-negative mutant, is present in Src-associated malignant transformation4,9. In general, intrinsic and extrinsic factors can stimulate tyrosine kinases, which phosphorylate Stat3 at tyrosine 705 (phospho-Stat3Y705) to generate binding sites for SH2 domain and in turn form homo- and heterodimers with Stat3 or other Stat members10. Activated Stat dimers then translocate to the cell nucleus, bind to specific DNA sequences and directly regulate expression of anti-apoptotic genes, including Bcl-xl and Mcl as well as pro-survival genes, like c-myc and cyclin D15,11. Phosphorylation at serine 727 (phospho-Stat3S727) contributes to achieve maximal activation of Stat312. Recently, several reports described the importance of phospho-Stat3S727, but not phospho-Stat3Y705, for the Stat3 mitochondrial translocation13,14. They showed that Stat3 in mitochondria interacted with enzymes of the electron transport chain (ETC) to regulate mitochondrial oxidative phosphorylation and facilitated Ras-induced malignant transformation13,15,16,17. There is also compelling evidence that increased levels of apoptotic cells have been frequently observed in Stat3 inactive or deficient tumor cells13,15,18. However, the signaling pathway involved in the lack of mitochondrial Stat3-mediated apoptosis is not well elucidated yet.

p38MAPK, ERK (extracellular signal-regulated kinase) and JNK (c-Jun NH2-terminal kinase) belong to the mitogen-activated protein kinase (MAPK) family. In comparison to ERK and JNK, which support cell proliferation and survival, p38MAPK has been widely accepted as an inhibitor of proliferation or a regulator of cell apoptosis14,19. p38MAPK can be phosphorylated and activated by diverse upstream activators MAPK kinase kinase (MKKKs), like ASK120,21,22,23. p38MAPK also acts as a free radical sensor and inhibits malignant transformation and tumorigenesis by inducing cell cycle arrest and apoptosis under oxidative stress18,23,24.

In this article, we studied the influence of Stat3-deficiency on cellular viability and found that Stat3-knockdown using small interfering RNA or CRISPR/Cas9 (referred to as KD cells) enhanced ROS-mediated apoptosis under oxidative stress. This synergistic effect was independent of phospho-Stat3Y705, but depended on p38MAPK activity. Chemical inhibition of p38MAPK or genetic repression of ASK1 led to rescue cellular damage. Interestingly, a similar rescue effect was observed by overexpression of Stat3Y705F in KD cells, but not Stat3S727D. In good agreement with previous results, we found that Stat3S727 is of importance for its localization in mitochondria. We showed that cells lacking functional Stat3S727 were more sensitive to oxidative stress, which depended on ASK1/p38MAPK. This connection between ASK1/p38MAPK signaling and mitochondrial Stat3-associated cellular apoptosis demonstrated by our data further support the notion that a specific mitochondrial Stat3 inhibitor could be of interest for clinical application.

Results

Stat3 knockdown leads to improved sensitivity to H2O2 in HeLa cells

Stat3 is present in most human cancer cells and is frequently activated by phosphorylation at Y705, which counteracts pro-apoptotic cascades and stimulate proliferation1. Recent reports indicated that phospho-Stat3Y705 is not the only modification and phospho-Stat3S727 also contributes to tumor cell proliferation under oxidative stress in certain cell lines13. To study the role of Stat3 in oxidative stress-related cellular proliferation, we depleted Stat3 in HeLa cells by transient transfection with Stat3 siRNA (thereafter referred to as HeLa siKD cells for knockdown cells and NC cells for negative control using non-targeting siRNA). The efficiency of knockdown was more than 70% detected by immunoblotting (Fig. 1A and densitometric analysis of Stat3 expression in SI. 1). An influence of the Stat3 knockdown on cell viability was hardly detectable in 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Sulforhodamine B (SRB) assay (Fig. 1B). However, upon 0.5 mM H2O2 the viability was dramatically reduced down to 40% in siKD cells, while 70% of living cells remained in NC cells (Fig. 1B).

Figure 1
figure 1

Synergistic toxic effect of H2O2 in combination with Stat3 siRNA. (A) The efficiency of siRNA knockdown in HeLa cells was analyzed by immunoblotting. WT: normal HeLa cells; NC: HeLa cells transfected with non-targeting siRNA. siKD: HeLa cells transfected with Stat3 siRNA. (B) HeLa NC and siKD cells were treated with H2O2 (0.5 mM) along, or in combination with diverse Stat3 inhibitors, including Stati (10 µM), Srci (1 µM) and E804 (1 µM) for 24 hrs. Then anti-proliferative effect was measured in MTT and SRB assays. Data were normalized to the value of non-treatment in WT cells showing the mean ± SD of quadruplicates and are representative of three independent experiments. (C) Inhibitory effects of Stat3 inhibitors were confirmed by immunoblotting using specific antibody against phospho-Stat3Y705 in HeLa cells. (D) Coefficient of drug interaction (CDI) was calculated for each combination as described in the experimental section. (E) NC or siKD cells were treated with increasing concentrations of H2O2 for 24 hrs. Annexin v/PI staining was performed to study cellular apoptosis. Apoptotic cells are annexin v-positive and necrotic cells are PI-positive. Living cells are double negative and late apoptotic cells are double positive. (F) Antioxidants prevented cell damage in MTT and SRB assays. NC or siKD cells were incubated with 0.5 mM H2O2 in combination with reduced glutathione (GSH, 5 mM) or N-acetyl-L-cysteine (NAC, 10 mM) for 24 hrs. (G) GSH and NAC rescued cell damage induced by H2O2 in apoptosis assay in siKD cells. (H) Elevated levels of ROS were detected in siKD cells exposed to 1 mM H2O2 for 1 hr and prevented by GSH. (I) Mitochondrial membrane potential was significantly reduced upon 1 mM H2O2 for 1 hr, which was compensated by adding GSH. The mitochondrial membrane potential is shown as the ratio of red-to-green floursecence stained by JC-1. + indicates in combination with H2O2. (***p < 0.001; **p < 0.01; *p < 0.05).

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Since phospho-Stat3Y705 contributes to cell proliferation11, we blocked the phospho-Stat3Y705 by recruiting three well-characterized Stat3 inhibitors, namely a Src inhibitor 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (Srci, Merck Src kinase inhibitor I), an in-house synthesized indirubin derivatives E80425,26,27,28 and the Stat3-SH2 domain inhibitor 2-hydroxy-4-(((-4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid (Stati, Merck Stat3 inhibitor VI). The concentrations of inhibitors were optimized to achieve a good inhibitory efficiency with a minimal toxicity (Fig. 1B,C). The inhibitory effects were confirmed by immunoblotting with a phospho-Stat3Y705 antibody (Fig. 1C and SI. 2). We compared the viability in HeLa NC or KD cells treated with H2O2 in the presence/absence of one of the inhibitors and calculated the coefficient of drug interaction (CDI, Fig. 1D and detail in method section). CDI showed a clear synergistic effect in the co-treatment of H2O2 with Stat3 siRNA (Fig. 1D), while an additive effect was observed in the combination with Stati, and slight antagonism in the presence of Srci and E804 (Fig. 1D).

Translocation of cytosolic phosphatidylserine (PS) from the inner to the outer leaflet of the menbrane is a hallmarker of apoptosis, which can be easily detected by fluorescent annexin v conjugate, while necrotic cells are propidium iodide (PI) positive because of the acute damage of cell membrane23,29,30. We treated siKD and NC cells with 0.25 mM and 0.5 mM H2O2 for 24 hr. We detected 7% and 14% annexin v positive cells in NC cells, while 17% and 37% in siKD cells (Fig. 1E and dot plots in SI. 3). Upon 1 mM H2O2 treatment only 30% cells are viable in both cell lines (Fig. 1E).

ROS are involved in cell death induced by H2O2 in Stat3-deficient HeLa cells

ROS are crucial in H2O2-induced cell death14,22. In agreement with this, anti-oxidants -either N-acetyl-L-cystein (NAC) or reduced glutathione (GSH)-, easily rescued H2O2-induced cell damage in both NC and siKD HeLa cells (Fig. 1F), the number of apoptotic cells (Fig. 1G and SI. 4).

Mitochondrial Stat3 contributes to the maintenance of the mitochondrial redox homeostasis during malignant transformation and proliferation15. Stat3-deficiency raised ROS formation from 5% in NC to 10% in siKD cells detected with dihydroethidium (DHE)23,29. Adding H2O2 increased ROS level to 22% in NC and 27% in siKD cells, which was neutralized by GSH (Fig. 1H).

Since aberrant ROS could be caused by the dysregulation of mitochondrial membrane potential23,29,31,32, we measured changes of membrane potential using 5,5′,6,6′-tetrachlorido-1,1′,3,3′- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a green fluorescent dye that can selectively enter into mitochondrial and at higher membrane potentials spontaneously forms red fluorescent J-aggregates23. Previous reports also showed that formation of the JC-1 dimer is suppressed under oxidative stress23,29. We found 21% reduction of the red-to-green ratio in NC cells upon hydrogen peroxide, which was further reduced in siKD cells (Fig. 1I). Adding GSH expectedly prevented membrane potential change (Fig. 1I). In summary, our results clearly demonstrated a pivotal role of ROS for enhancing apoptotic effect mediated by H2O2 in Stat3-deficienct HeLa cells.

ASK1 activated p38MAPK contributes to the synergistic effect of H2O2 induced apoptosis

Recently, several reports indicated that excessive ROS can activate p38MAPK-associated apoptotic cascades18,20,21,22,23. In comparison to NC cells, we detected increased levels of phospho-p38MAPK in siKD cells exposed to H2O2 for 1 hr visualized either by immunocytochemistry (Fig. 2A) or immunoblotting (Fig. 2B and SI. 5), while total p38MAPK remained constant (Fig. 2B). Recently we showed that the inactivation of ASK1 can abolish p38MAPK-mediated cellular apoptosis induced by Trx-R inhibitor23. To investigate if the ASK1/p38MAPK cascade plays a role in this context, we employed SB203580, a well-known p38MAPK inhibitor (p38i), or ASK1 siRNA (siASK1) published before23. The combination led to the reduction of H2O2-induced activation of p38MAPK in siKD cells (Fig. 2C and SI. 6). We evaluated the impact of p38i and siASK1 on H2O2 mediated cell damage using trypan blue (Fig. 2D), MTT (Fig. 2E), SRB (Fig. 2E) and apoptosis assay (Fig. 2F). The results consistently showed that the inhibition of p38MAPK or ASK1 knockdown could effectively prevent cell death in siKD cells. Moreover, PARP and caspase 3 are hallmarks of apoptosis33. We examined the cleavage of both in NC and siKD HeLa cells treated with H2O2 for 24 hrs in presence or absence of p38i. The results clearly show the enhanced cleavages of PARP and Caspase 3 in siKD cells (Fig. 2G and SI. 7), which could be prevented by p38i (Fig. 2G).

Figure 2
figure 2

ASK1/p38MAPK signaling pathway plays an important role for inducing synergistic effect in Stat3-deficient HeLa cells under oxidative stress. (A) p38MAPK was activated in siKD cells in the presence of 1 mM H2O2 for 1 hr. Phospho-p38MAPK was stained with pp38MAPK antibody as green and nuclei were visualized with DAPI as blue. (B) Activated p38MAPK was detected in siKD cells treated with H2O2. Cells (wt, NC or siKD) were treated with two concentrations of H2O2 for 1 hr. Whole cell lysates were subjected to immunoblotting and analyzed for phospho-p38MAPK, total p38MAPK and Stat3 using respective antibodies. ß-actin was used as loading control. 40 µg proteins were loaded per lane. (C) ASK1 siRNA (siASK1) and p38MAPK inhibitor (p38i) reduced phosphorylation of p38MAPK. Cells were transfected with Stat3 and ASK1 siRNA for 48 hrs. Non-targeting siRNA was used as control in NC cells. Cells were incubated with H2O2 in absence or presence of p38i for 1 hr. Whole cell lysates were analyzed by immunoblotting for expression of phospho-p38MAPK, ASK1, Stat3 and ß-Actin. (D) and (E) p38i or siASK1 increased cell survival. Cells were treated with 0.5 mM H2O2 in combination with p38i or siASK1 for 24 hrs. The cell growth was measured using Trypan blue (D), MTT (E) and SRB (E) assays. The data were normalized to non-targeting siRNA control, showing the mean ± SD of quadruplicates and are representative of three independent experiments. (F) p38i or siASK1 prevented cell apoptosis. Cells were treated with p38i or siASK1 and analyzed using annexin V/PI staining. (G) NC or siKD cells were treated with compounds as indicatea in the text. Whole cell lysates were analyzed for PARP, Caspase 3 and Stat3 levels. + indicates in combination with H2O2. (***p < 0.001; **p < 0.01; *p < 0.05).

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Phospho-S727 Stat3 localizes in mitochondria

Recently, Larner and Levy groups described that phospho-Stat3S727, but not phospho-Stat3Y705, led to the accumulation of Stat3 in the mitochondrion13,15. They also showed that mitochondrial Stat3 contributed to the activity of ETC as well as to tumor initiation and progression13,15. Our above results showed that the suppression of phospho-Stat3Y705 by chemical inhibitors was not able to mimic the effect arising in KD cells. We hypothesized that mitochondrial Stat3 might play a crucial role in our observed ASK1/p38MAPK-mediated KD cell apoptosis. Hence we prepared cytosolic and mitochondrial extracts from HeLa cells and compared levels of total Stat3, phospho-Stat3Y705 and phospho-Stat3S727 (Fig. 3A). In agreement to previous results15, only a trace of mitochondrial Stat3 (10% in comparison to cytosolic Stat3) was detected in HeLa cells, while phospho-Stat3Y705 was undetectable in mitochondria. The level of phospho-Stat3S727 in mitochondria was comparable to that in cytoplasm (Fig. 3B), suggesting a crucial role of phosphorylation at S727 for localization of Stat3 in mitochondria15.

Figure 3
figure 3

Loss of functional mitochondrial Stat3 is essential to enhance cell sensibility to oxidative stress. (A) and (B) Mitochondrial and cytosolic fractions were isolated from HeLa cells to compare the concentrations of Stat3, phospho-Stat3Y705 and phospho-Stat3S727 by immunoblotting in six independent experiments. Signal intensities were quantified and normalized to corresopnding proteins in the mitochondrial fraction. ß-actin and COX IV were used for loading control. (C) Schematic illustration: the generation of CRISPR/Cas9 mediated Stat3 knockdown cell lines (HeLa cKDs) with signle cell expansion. (D) The knockdown effect of HeLa cKDs. (E) GSH, ASK siRNA (siASK) and p38 inhibitor (p38i) sufficiently blocked the synergistic toxic effect by H2O2 in cKD5 cells measure by MTT and SRB assays. (F) Coefficient of drug interaction (CDI) was calculated. (G) Overexpression of Stat3Y705F, but not Stat3S727D rescued H2O2-induced ROS formation in cKD5 cells. (H) Adding p38 inhibitor (p38i) and overexpression of Stat3Y705F, but not Stat3S727D prevented cellular apoptosis induced by H2O2 in cKD5 cells. (I) Overexpression of Stat3Y705F, but not Stat3S727D blocked PARP cleavage induced by H2O2 in cKD5 cells. (J) Adding p38 inhibitor (p38i) sufficiently inhibited phospho-p38MAPK and cleavage of PARP induced by H2O2 in cKD5 cells with/without overexpression of Stat3S727D. (K) Localization of Stat3 in HeLa cells expression various genetic modified Stat3. M: mitochondrial Stat3, C: cytosolic Stat3. HSP60 and Vincublin was used as marker for mitochondrion and cytosol respectively. (L) Co-localization of Stat3 (red) and mitochondrial marker HSP60 (green) in HeLa WT and Stat3Y705F, but not Stat3S727D, using confocal microscope. The expression level of Stat3 in cKD cells is undetectable. Hoechst dye was used to indicate nuclear. Arrow: co-locolization of Stat3 and HSP60. Scale bar: 10 µm. + indicates in combination with H2O2. (***p < 0.001; **p < 0.01; *p < 0.05).

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Stat3S727 plays an essential role in p38MAPK-mediated apoptosis

We generated HeLa Stat3 KD cell lines using a commercially available CRISPR/Cas9 plasmid. A number of studies indicate that off-target effect of Cas9 depends on both sgRNA sequence and experimental conditions34,35. To obtain genetically homologous knockdown cell lines, we established 10 HeLa Stat3 CRISPR/Cas9 KD cell lines (cKDs) using single cell expansion as illustrated in Fig. 3C (detailed description in the method section). The knockdown effect was examined by immunoblotting (Fig. 3D). Among them, cKD5 showed the best knockdown effect (70%, SI. 8 and 9) and therefore was selected for our further experiments.

In good agreement to published findings13,15, we found less mitochondrial activity in cKD cells than in NC cells by measuring the oxygen consumption (SI. 10)36. We performed MTT assay (Fig. 3E), SRB assay (Fig. 3E), calculated CDI (Fig. 3F), measured ROS formation using DHE and Mitosox (Fig. 3G and SI. 11), compared mitochondrial membrane potential detected by TMRE (SI. 12) and quantified apoptotic cells (Fig. 3H and SI. 13) in cKD5 cells, and also monitored the mitochondrial ROS by adding Mitosox in real-time (Video 1–7). The results obtained were similar to those in KD cells, confirming the lost ability to regulate the cellular redox balance of Stat3-deficient cells, which led to activation of the ASK1/p38 cascade and consequently to apoptosis under oxidative stress (Fig. 3E,H–J and SI. 14 and 15).

To test the function of mitochondrial Stat3 in this context, we overexpressed Stat3 with either S727D mutation (Stat3S727D) or Y705F mutation (Stat3Y705F) in cKD5 cells. The results show that excessive ROS formation under oxidative stress could be compensated by ectopic expression of Stat3Y705F, but not Stat3S727D (Fig. 3G). As a result, the reduction of annexin v positive cells (Fig. 3H and SI. 13) and PARP cleavage (Fig. 3I and SI. 14) was observed, showing a p38-dependend manner (Fig. 3I,J). To confirm mitochondrial localization, we purified mitochondria from cells and directly analyzed the levels of mitochondrial Stat3, which confirmed that S727 was essential for mitochondrial location of Stat3 (Fig. 3K and SI. 16). Moreover, immunostaining with anti-Stat3 antibody (Fig. 3L, red) and mitochondria specific anti-HSP60 antibody (Fig. 3L, green) showed a co-locolization of Stat3 and HSP60 in mitochondria in HeLa wt, but not Stat3S727D, while Stat3 was undetectable in cKD5 cells (Fig. 3L). The signal became much clearly in the case of Stat3Y705F.

Stat3-deficiency related p38MAPK activation in HEK293

Next we tested if the synergistic effect appears in HEK293 cells using Stat3 siRNA (HEK293 siKD). As shown in Fig. 4A and SI. 17, 20% annexin v positive cells could be detected in NC cells upon 0.5 mM H2O2, while 70% were found in HEK293 siKD cells. The calculated CDI was comparable to that in HeLa KD cells (Fig. 4B). Analyzing the level of Stat3 expression and activity of p38MAPK by immunoblotting, we confirmed the suppressed Stat3 expression and activation of p38MAPK in HEK293 siKD cells after exposure to H2O2 (Fig. 4C).

Figure 4
figure 4

The synergestic effect is reproducible in Stat3-deficient HEK293 cells and in HeLa KD cells under hypoxia. (A) Enhanced apoptotic effect was observed in HEK293 transfected with Stat3 siRNA. Cells were treated with H2O2 for 24 hrs and analyzed by apoptosis assay. Apoptosis assays were performed following treatment. (B) Coefficient of drug interaction. (C) Stat3 deficiency contributed to hyperactive phosphorylation of p38MAPK in the presence of H2O2. Cells were treated with 1 mM H2O2 for 1 hr. The whole cell lysate was subjected to immunoblotting. (D) Cells were counted after 24 hrs treatment with or without p38i in HeLa siKD cells under normoxic or hypoxic using trypan blue assay. Nor: normoxia; Hyp: hypoxia. (E) MTT assay was performed to evaluate the anti-proliferative effect of small molecules (p38i) or ASK siRNA (siASK) as indicated under hypoxia in HeLa siKD cells. (F) The influence of p38i and siASK on cell apoptosis under hypoxia was measured by annexin v/PI assay. (**p < 0.01; *p < 0.05).

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siKD HeLa cells are more sensitive to hypoxia

To investigate whether this synergistic effect is reproducible under hypoxia, we cultivated HeLa cells under 1% oxygen13 and analyzed the cellular damage by trypan blue staining (Fig. 4D), MTT (Fig. 4E) and apoptosis assays (Fig. 4F and SI. 18) as described above. The viability of NC cells under normoxia and hypoxia was similar, which was reduced to 60% in siKD cells under hypoxia (Fig. 4D,E). Combination with p38i or siASK1 significantly increased cell survival up to 80% (Fig. 4D,E), in good agreement with our former results that inhibition of ASK1/p38MAPK cascade prevented the synergistic effect under oxidative stress in the absence of Stat3. FACS analysis also confirmed that hypoxia induced more than 50% apoptotic cells in siKD cells, which was clearly inhibited by p38i or siASK1 (Fig. 4F).

Discussion

The evidence that most of liver and gastric tumors originate from chronic inflammation commonly induced by infections with hepatitis B virus and hepatitis C virus, supports the concern that inflammation facilitates and promotes tumor initiation and progression3. In spite of the involvement of numerous genes and proteins, Stat3, a transcription factor, has been identified as an essential element in inflammation-associated carcinogenesis. Thus, the strategy to target Stat3 signaling may be beneficial for cancer therapy5,9,37. However, the failure to develop Stat3 inhibitors for clinical use implies that a deeper understanding of the molecular basis of Stat3 signaling is required38.

In the present study, we found that knockdown of Stat3 using siRNA or CRISPR/Cas9 enhanced apoptotic effect under oxidative stress induced by either H2O2 or hypoxia. In general, active Stat3 is associated with tumor cell proliferation1,8. In the canonical Stat3 cascade, phospho-Stat3Y705 is important for Stat3 translocation into nuclei to regulate downstream gene expression1,23,39,40. We selected three structural distinct inhibitors of canonical Stat3 signaling, but none of them could synergistically inhibit cell growth in combination with H2O2. Apparently, transcription activity of Stat3 is not involved in the synergistic effect of apoptosis induced by oxidative stress in cells lacking Stat3.

Recently, Wegrzyn et al. reported the identification of a trace of phospho-S727 Stat3 in mitochondria, which plays an important role in the maintenance of mitochondrial activity and suppression of ROS release from the ETC15,41. Moreover, mitochondrial Stat3-mediated accumulation of ROS promoted breast cancer growth42. In good agreement, our results showed accumulation of ROS levels and reduction of mitochondrial membrane potential in KD cells. We detected abundant phospho-Stat3S727 in the mitochondrial fraction. Overexpression of StatY705F in KD cells neutralized excessive ROS and rescued cellular apoptosis in KD cells, but not Stat3S727D, which implicated an important role of functional S727 in regulation of cellular redox homeostasis.

MAPKs signaling is one of the most well-understood signaling processes involved in tumorigenesis43,44,45. In comparison to other MAPKs, whose functions in cell growth are still controversial, p38MAPK has been identified mostly as a pro-apoptotic protein19,46,47,48, which can be activated by inflammation, environmental and genotoxic stresses49,50. In Ras driven cell transformation, p38MAPK can be activated by increased ROS and in turn attenuate malignant transformation, while inhibition of p38MAPK leads to ROS abundance and initiation/progress of tumor19,51. In KD cells, a clear hyperactive p38MAPK was detected. Inhibiting p38MAPK with chemicals or anti-oxidants interfered with peroxide-induced cell death, suggesting that the synergistic toxic effect is associated with ROS-mediated active p38MAPK.

ASK1 has been described as an activator of p38MAPK involved in cell apoptosis induced by ROS22,52. Under oxidative stress, binding of Trx to ASK1 is disrupted by oxidization of Trx leading to the release and thereby activation of ASK1. ASK1 can then phosphorylate MKK3 and its downstream kinase p38MAPK to induce cell apoptosis19,23,51,53. As expected, knockdown of ASK1 by using siRNA not only reduced the level of phospho-p38MAPK, but also protected cells from damage under oxidative stress and mimicked the effect of p38i in KD cells, supporting our hypothesis that Stat3-deficiency acts synergistic with the ASK1/p38MAPK cascade in the induction of apoptosis.

Taken together, our results demonstrate that knockdown of Stat3 conferred cells more sensitivity under oxidative stress, most likely due to the lack of mitochondrial Stat3, which is of importance to maintain mitochondrial activity. Knockdown of Stat3 significantly reduced the malignant transformation and tumor proliferation, and induced cell damage in vitro and in vivo13,15,41. However, little is known about the associated molecular targets. Our findings clearly indicate that apoptosis associated with the lack of mitochondrial Stat3 was mediated by the ASK1/p38MAPK cascade. Our results imply that the development of inhibitors targeting the mitochondrial activity of Stat3 could be of clinical interest as potential anti-tumor agents and could work either by interference with phosphorylation at S727 or by targeting Stat3 for degradation.

Materials and Methods

Materials

E804 was synthesized as previously described25. Structure and purity were ascertained by13C- and1H-NMR spectroscopy and elemental analyses. N-acetyl-L-cysteine (NAC), glutathione (GSH) and p38 inhibitor (p38i, SB203580) were purchased by Sigma-Aldrich (Germany). p38 (cat: #9212), pp38 (T180/Y182, cat: #9215), Stat3, pStat3 (Y705), pStat3 (S727), Caspase 3 and COX IV were obtained from cell signaling (NEB, Germany). ASK1 (cat: sc-7931) and Actin were from Santa Cruz (Germany).

Cell culture

HeLa and HEK293 cells were cultivated in DMEM with 10% FBS and 1% Pen/Strp (PS) under 5% CO2 at 37 °C in a humidified atmosphere and treated with compounds as indicated. siRNA oligonucleotides were synthesized by riboxx as previously reported54. Sequence of ASK1 siRNA (siASK1) was reported previously23. Riboxx®FECT reagent was used to increase the transfection efficiency. After 48 h cells were incubated with compounds as indicated. Hypoxia (1% O2) was generated by Thermo Scientific Heraeus® Cytoperm® CO2/O2 Incubator as reported29, The cells were plated in normoxia condition for 24 hrs prior to transfection and were immediately placed in hypoxia incubator after transfection. All of the treatments were conducted within 1 hr after 48 hrs transfection and re-placed in hypoxia condition directly after treatment. All the assays were performed 24 hrs later in normoxia condition. CRISPR/Cas9 knockdown cell line was generated by using commercially available CRISPR/Cas9 plasmid (Santa Cruz, Germany). The transfection was performed as previously described using Lipofectamine 3000 (Life Technologies, Germany). 1 µg/mL puromycin was used to select transfected cells. Three days later, 10 colonies were selected manually under microscope and each was re-plated in a well of 6-well plate for 5 days. 10–100 cells of each sub-colony were isolated by using a plastic scraper. After re-plating with single cell suspension, medium was changed after 3 hrs. The places seeded with single cells or after removal of other cells were marked under microscope. The cell lines from each colony developed from single cells were isolated and established in medium without puromycin. Stat3S727D (Addgene 73364) and Stat3Y705F (Addgene 74434) plasmids were reported previously55 and purchased from Addgene. Isolation of mitochondrial and cytosolic Stat3 was described as previously reported56.

Trypan blue assay

The cells were cultivated in DMEM (10% FBS), transfected with siRNAs and treated with compounds. The cells were trypsinized and re-suspended in medium. A mixture of 20 µL cell suspension and 20 µL trypan blue was added into a hemocytometer chamber29. The number of cells was scored under microscopy. The absolute values were listed.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) and Sulforhodamine B (SRB) assay

MTT and SRB assays were performed to determine anti-proliferative effect of compounds as reported23. Briefly, after 24 hrs seeded in 96-well plates, the transfection was performed for 48 hrs in DMEM containing 10% FCS and thereafter the cells were treated for 24 hrs in quadruplicate. The medium was removed and a solution of MTT was added for MTT assay, incubated for 1 h and quantified photometrically at 560 nm in DMSO. For SRB assay, trichloroacetic acid (50% solution) was added to stop the incubation (1 h at 4 °C). Plates were washed with water and dried overnight. A solution of 0.4% sulforhodamine B was added to stain proteins and washed with 1% acetic acid. The dye was solved with Tris buffer (10 mM, pH = 10.5) and quantified photometrically at 560 nm.

ROS Formation assay

Cells were trypsinized, centrifuged at 200 g (1500 rpm), resuspended in FACS buffer containing 1% BSA in D-PBS and incubated with 5 µM dihydroethidium (DHE, Sigma-Aldrich), or Mitosox at room temperature in the dark for 15 min and then immediately analyzed by Fluorescence-activated cell sorting (FACS) using a FACSCalibur (Becton Dickinson) and CellQuest Pro (BD) analysis software. Excitation and emission settings were 488 nm and 564–606 nm (FL2 filter), respectively29.

Apoptosis assay

Cells were cultured in DMEM, transfected with siRNAs for 48 hrs and incubated with compounds. The harvested cells were resuspended in 50 µL annexin V binding buffer, incubated with 5 µL FITC-conjugated annexin V (BD Bioscience, Germany) for 15 min in the dark at room temperature. Afterwards, 450 µL annexin V binding buffer containing 1.25 µL propidium iodide (PI, 1 mg/mL) were added, and incubation continued for 10 min in the dark at room temperature before analyzis by FACS29.

Membrane potential measurement and oxygen consumption

Cells were incubated with 500 nM JC-1 (5,5,6,6-tetrachloro-1,1,3,3- tetraethylbenzimidazolylcarbocyanineiodide, Sigma-Aldrich) or 100 nM TMRE (Santa Cruz) for 15 min at 37 °C. Afterwards, cells were harvested and analyzed by FACS. Excitation and emission settings were 488 nm, 515–545 nm (FL1 channel) for JC monomers, and 564–606 nm (FL2 channel) for JC aggregates23. The oxygen consumption measurement was performed as our previously reported36.

Immunoblotting and immunostaining

Cells were collected in urea-lysis buffer (1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 6 M Urea, 1 mM Na3VO4, 10 µg/mL Pepstatin, 100 µM PMSF and 3 µg/mL Aprotinin in PBS). Enhanced chemiluminescence (ECL) immunoblotting analysis was used and 40 µg of total protein were resolved on 8–10% SDS-PAGE gels and immunoblotted with specific antibodies as described previoiusly57,58.

Imaging

Confocal fluorescent images were acquired using a TSC SP5 confocal microscope (Leica), a HCX PL APO 63x/1.3 GLYC CORR objective and LAS5 microscope imaging software (Leica). For image processing, Fiji/ImageJ was used.

Evaluation of combinatory effects

Based on the method for calculation the coefficient of drug interaction (CDI) the combined effects of Stat3-depletion and oxidative stress were calculated. The effect of combination was calculated as follows: CDI = AB/(A × B), in which AB indicates viability of the combination; A and B indicate cell viabilities of the respective single treatment. CDI < 1 indicates a synergistic effect, CDI < 0.7 indicates a significant synergistic effect; CDI = 1 an additive effect; CDI > 1 an antagonistic effect.

Statistical analysis

The statistical significance of compared measurements was performed using the Student’s one-tailed or two-tailed t-test (Microsoft Excel).

All data generated or analysed during this study are included in this published article.