STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells - PubMed (original) (raw)

STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells

Sevda Jafari et al. Daru. 2020 Jun.

Abstract

Background: Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. Stattic is an inhibitor of STAT3, which is found constitutively active in many cancers and plays a major role in cancer progression.

Objectives: In the present study, we proposed to evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3.

Methods: The growth inhibitory effects of stattic and chemo agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box 1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA.

Results: Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, we found a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, we did not observe a significant increase in the level of ICD in CT26 cells and IL-12 secretion by DCs when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups.

Conclusion: These findings indicate that STAT3 inhibitory stattic can increase ICD induced by DOX. Graphical abstract.

Keywords: Chemotherapy; Colon cancer; Combination therapy; Immunotherapy; Melanoma; STAT3.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

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Graphical abstract

Fig. 1

Fig. 1

Dose-response curves of (a) stattic and (b) DOX in B16F10 cells, (c) stattic, (d) DOX and (e) OXP in CT26 cells. Dose-response curves were generated by GraphPad prism software to determine IC50 value of the drugs. Growth inhibitory effects of each monotherapies and combination therapies were obtained from MTT assay (f) DOX, stattic and DOX + stattic in B16F10 cell, (g) DOX, stattic and DOX + stattic and (h) OXP, stattic and OXP + stattic in CT26 cells. The data represent mean ± SD (n = 3). Representative western blotting analysis of p-STAT3, STAT3 and B-Actin in (i) B16F10 cells treated with DOX, stattic and DOX + stattic, (j) CT26 cells treated with DOX, stattic, OXP, DOX + stattic and OXP + Stattic. DOX, doxorubicin; OXP, oxaliplatin

Fig. 2

Fig. 2

Representative dot plot graph showing frequency of early and late apoptotic death after 48 h treatment of (a) B16F10 cells with DOX, stattic and DOX + stattic and (b) CT26 cells with DOX, OXP, stattic, DOX + stattic and OXP + stattic, as assessed by Annexin/PI staining and analyzed by flow cytometry. Each experiment was conducted in triplicate. DOX, doxorubicin; and OXP, oxaliplatin

Fig. 3

Fig. 3

Representative histogram of CRT surface exposure in (a) B16F10 cells treated with DOX, stattic and DOX + stattic, and (b) CT26 cell treated with DOX, OXP, stattic, DOX + stattic and OXP + stattic. Staining was assessed by PE-conjugated anti-CRT antibody and the experiments were analyzed by flow cytometry. The data represent one out of three independent experiments, which showed similar results. DOX, doxorubicin; OXP, oxaliplatin and PE, phycoerythrin

Fig. 4

Fig. 4

Assessment of HMGB1 and HSP70 release by ELISA following treatment with (a) DOX, stattic and DOX + stattic in B16F10, (b) DOX, stattic and DOX + stattic and (c) OXP, stattic and OXP + stattic in CT26 cells. The experiments were repeated in triplicate and data are indicated as the mean ± SD. DOX, doxorubicin; OXP; oxaliplatin, ns; not significant

Fig. 5

Fig. 5

IL-12 secretion in DCs assessed by ELISA following the exposure to CM obtained from (a) B16F10 cells, (b) and (c) CT26 cells. Experiments were repeated in triplicate and data are indicated as the mean ± SD. DOX, doxorubicin; OXP, oxaliplatin; not significant, ns

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