Artesunate promotes sensitivity to sorafenib in hepatocellular carcinoma

Biochemical and Biophysical Research Communications 519 (2019) 41e45

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Artesunate promotes sensitivity to sorafenib in hepatocellular carcinoma Wu Jing a, b, c, *, 1, Liu Shuo a, 1, Xing Yingru d, Mu Min b, Zhao Runpeng a, Xie Jun d, Hu Dong a, b, c, * a

Department of Medical Immunology, Anhui University of Science and Technology, Huainan, PR China Key Laboratory of Industrial Dust Prevention and Control & Occupational Safety and Health of the Ministry of Education, Anhui University of Science and Technology, Huainan, PR China c Key Laboratory of Industrial Dust Deep Reduction and Occupati onal Health and Safety of Anhui Higher Education Institute, Anhui University of Science and Technology, Huainan, PR China d Affiliated Cancer Hospital, Anhui University of Science and Technology, Huainan, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2019 Accepted 22 August 2019 Available online 31 August 2019

Enhancing sensitivity of carcinoma to sorafenib (Sor) is critical to overcome the limits of high frequency resistance and moderate efficiency during chemotherapy for advanced hepatocellular carcinoma (HCC). Here, we promote sensitivity of HCC to Sor by combination with Artesunate (Art), a derivative of artemisinin extracted from Chinese medical herb. The positive synergy of Art on inhibiting HCC growth contributes 48% dosage of Sor to reduce tumor cell viability in vitro and tumor size in vivo. Mechanically, in spite of effective suppression of RAF/MEK/ERK pathway, Sor is not able to eliminate chemoresistance of HCC driven by PI3K/AKT/mTOR pathway, while Art inhibits phosphorylation of AKT and mTOR significantly. Furthermore, combination with Art and Sor further improves apoptosis of HCC by dual inhibition of both pathways. Our study reveals a function of Art that induces HCC apoptosis via PI3K/AKT/ mTOR pathway inhibition and suggests a potential therapeutic regimen of combination with Art and SOR against advanced HCC. © 2019 Elsevier Inc. All rights reserved.

Keywords: Hepatocellular carcinoma Sorafenib Artesunate PI3K/AKT/mTOR RAF/MAPK Apoptosis

1. Introduction Sor is a tyrosine kinase inhibitor approved for the clinical treatment of advanced primary liver cancer generally by blocking RAF/MAPK (mitogen-activated protein kinase), VEGFR (vascular endothelial growth factor receptor) and PDGFR (platelet-derived growth factor receptor) kinases [1]. However, due to the high heterogeneity of HCC, multiple pathways were activated and drove progression, chemoresistance and metastasis of HCC [2]. Among of which, PI3K/AKT/mTOR is a major pathway activated in 30%e50% HCC cases [3] and can be induced by Sor treatment [4]. Art is a semi-synthetic derivative of artemisinin as the first line treatment for malaria [5]. Besides that, Art shows anti-tumor

* Corresponding authors. Chongren Building, No 168, Taifeng St, Huainan, 232001, PR China. E-mail addresses: [email protected] (W. Jing), [email protected] (H. Dong). 1 These authors contribute equally to this study. https://doi.org/10.1016/j.bbrc.2019.08.115 0006-291X/© 2019 Elsevier Inc. All rights reserved.

effects in various ways, including cell cycle arrest, apoptosis, autophagic death and EMT block [6e8]. More importantly, PI3K/ AKT/mTOR is a major pathway targeted by Art in a variety of cancers, by which Sor shows a high frequency chemoresistance and moderate efficacy in clinical application [9,10]. Therefore, it is suggested that Art may overcome chemoresistance of HCC to Sor by suppression of PI3K/AKT/mTOR pathway. In this study, we have shown that Art promotes apoptosis of HCC which correlated to reduced PI3K/AKT/mTOR pathway. Combined with Sor, Art also blocks RAF/MAPK pathway and enhances the therapeutic efficacy of Sor against HCC in vivo and in vitro. 2. Results To investigate whether Art enhanced anti-tumor effect of Sor, we treated node mice herbing SK-7721 tumor cells with saline, Sor, Art or Sor combining Art (Fig. 1A). Both Sor and Art reduced tumor growth as decreased tumor size and tumor weight (Fig. 1B and C). Whereas, combined treatment with Sor and Art further reduced tumor growth compared to treatment with Sor or Art alone (Fig. 1B and C).

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Fig. 1. Art inhibits tumorigenesis and enhances chemotherapeutic effects of Sor in mice HCC models A: representative images for in-vivo tumors in and isolated tumors from mice treated with DMSO (Con), 2.5 mg/kg sorafenib (Sor), 100 mg/kg artesunate (Art), Sor (2.5 mg/kg) combined with Art (100 mg/kg) by i.p. i njection every other day for 4 weeks (n ¼ 6). B: Tumor growth curve using tumor size by the volume formula p/6(L  W  H). Mean ± SEM, *p < 0.05; **p < 0.01 by Welch’s t-test, n ¼ 6. C: Tumor weight analysis. Mean ± SEM, ***p < 0.001; ****p < 0.0001 by Welch’s t-test, n ¼ 6.

To further understand how Art improves Sor chemotherapeutic sensitivity, we analyzed the effect of Sor on SK and SM live cancer cells in vitro. Both in SK and SM cells, IC50 of Sor and Art was examined (Fig. 2A and B), whereas, isobologram curve of combined treatment fell in the left of fitting curve (Fig. 2C and D). In other words, 2.77 mM Sor combined with Art was equal to 5.23 mM Sor alone for 50% inhibition of SK and 11.43 mM Sor combined with Art equal to 5.30 mM Sor alone for SM. These data suggested there is a positive synergy between Sor and Art to inhibit live cancer cells. To validate this, cell count assay also showed that combined treatment dramatically reduced the alive cell numbers both in SK and SM compared to Sor or Art alone (Fig. 2E and F). To investigate how Art promotes Sor chemotherapeutic efficacy, we analyzed the cellular apoptosis. Combined treatment significantly increased annexin Vþ SK cell population in flow test, compared to treatment with Sor or Art alone (Fig. 2H and I). Furthermore, incorporating Art significantly increased cleaved caspase 3 and cleaved PARP as markers of apoptosis (Fig. 2J). Collectively, our results suggest that Art positively synergize Sor to inhibit liver tumor cell by pro-apoptotic processes. To identify the signaling pathways activated by Sor and Art, we analyzed the PI3K/AKT/mTOR and RAF/MAPK pathways using Western blot assay (Fig. 3). Consistent to previous studies [6,11], we also found a high expression of phosphor-RAF1(a MAP3K), -ERK1/2 (a MAPK) and phosphor-AKT, mTOR in SK tumor cells, suggesting both MAPK and PI3K/AKT/mTOR pathways were active in SK cells. However, sore inhibited the MAPK pathway as reduced phosphorRAF1 and -ERK1/2 but not PI3K/AKT/mTOR pathway, whereas, Art inhibited PI3K/AKT/mTOR but not MAPK pathway. Furthermore, combined treatment with Sor and Art suppressed phosphorylation of RAF, ERK, AKT and mTOR, suggesting multiple key enzymes in both pathways were blocked by this combination. Of note, we observed treatment with Rapamycin alone induced a suppression of phosphor-mTOR, but when combination the inhibitory effect lost either in p-RAF1 and -ERK1/2 by Sor or p-mTOR by rapamycin, suggesting there was a negative synergy between rapamycin and Sor.

To analyze the correlation between apoptosis and pathways activated in liver tumor, we examined the expression of an apoptosis marker (cleaved PARP) and key enzymes in MAPK (phosphor-ERK) and PI3K/ARK/mTOR (phosphor-mTOR) pathways using immunofluorescence assay (Fig. 4A and E). Combined treatment with Art significantly reduced the ratio of p-mTOR positive tumor cells (Fig. 4A and B) but promoted c-PARP positive cells (Fig. 4A and C), whereas, a negative correlation was found between them (Fig. 4D). Similarly, combined treatment with Art induced a decreased proportion of p-ERK positive tumor cells (Fig. 4E and F) accompanying an increased abundance of p-PARP positive cells (Fig. 4E and G), and their number were negatively associated (Fig. 4H). Thus, it was verified in vivo that the combination with Sor and Art improved tumor cell apoptosis by the enhanced inhibition in both MAPK and PI3K/AKT/mTOR pathways. 3. Discussion In this study, we show that both RAF/MAPK and PI3K/AKT/ mTOR pathways highly activated in HCC were impinged by combination treatment with Sor and Art, by which apoptosis was dramatically raised and impelled a markable inhibition of HCC in vitro and in vivo. Tumor recurrence has been a major issue for Sor treatment in advanced HCC [12]. Currently, specific surface markers are lacking to accurately predict the recurrence induced by resistance to Sor in many tumor types [13]. However, in patients with long-term monotherapy of Sor, drug resistance is associated with highly expressed enzymes composed PI3K/AKT/mTOR pathway [14,15]. Therefore, recently increased researches have made effort to overcome this issue and proved various combination therapies with PI3K inhibitors [2,16]. Previously, we reported Art inhibited nasopharyngeal carcinoma cells by blocking PI3K/AKT/mTOR pathway [9]. Here, we found HCC cells were characterized by double pathways of PI3K/AKT/mTOR and RAF/MAPK, consisting to other reports [3,11,16]. Based on it, we combined Sor with Art, by which both pathways were significantly suppressed. Consistently,

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Fig. 2. Art promotes sorafenib to inhibit proliferation and induce apoptosis in vitro SK or SM cells were treated with DMSO (con), Sor, Art or Sor combined with Art (1:50). (A, B) IC 50 curves were fitted in SK and SM cells using least square method. IC50 of Sor (A) at doses (0.316e 100 mM) and Art (B) at doses (1e1000 mM) in SK and SM cells were calculated. (C, D) Viability inhibition curves were fitted using least square method. Positive synergistic curves (purple bold solid line) and fitting synergistic curves (purple bold dashed line) for combination with Sor and Art were figured out in SK (C) and SM (D) cells. (EeG) Cell counts using trypan blue staining. Representative image (E) was shown and analyzed for living SK (F) and SM (G) cells treated with DMSO, Sor (1 mM), Art (50 mM), Sor combined with Art (1:50). n ¼ 6. (H) Flow cytometric analysis of annexin V and PI stained SK cells with treatments for 24 h at doses as E, apoptosis cells were analyzed (I). n ¼ 3. (J) Western-blot analysis of cleaved-CASP3, cleaved-PARP and ACTB in SK cells with treatments for 24 h at doses as E. (F, G, I) Mean ± SEM, **p < 0.01; ***p < 0.001; ****p < 0.0001 by Welch’s t-test.

we found the combination induced further increase of apoptosis that negatively related to the key components of the two pathways. However, a different effect was found if combined with mTOR inhibitor (rapamycin), as the two pathways were not decreased but increased compared to single treatment with either Sor or rapamycin, suggesting an absence of synergy between them and a partial explanation for the absence of effect on apoptosis [17]. In summary, our study identifies a positive synergistic effect between Sor and Art on HCC treatment, as it promotes a dual inhibitory effect on both RAF/MAPK and PI3K/AKT/mTOR pathways which contributes to apoptosis burst. Of note is that whether this combination targeting both pathways may represent a more precise therapy of HCC warrants further clinical investigation.

4. Material and methods 4.1. Mice models and tumor samples

Fig. 3. Art promotes sensitivity to sorafenib in SK cells by inhibiting PI3K/mTOR and RAR/ERK pathways. SK cells were treated with Sor (1 mM) or Art (50 mM) alone or combination of Sor and Art (1:50) with or without Rapamycin (5 mM) for 5 h. Representative western blots showing phospho-c-Raf (Ser338), Phospho-ERK1/2 (Thr202/Tyr204), phosphoAKT(Ser473), phospho-mTOR (Ser2448) and GAPDH as internal control.

All procedures related to animals were performed according to the Guide for the Care and Use of Laboratory Animals (NIH publication no. 80-23, revised 1996) and approved by the medical ethics committee of Anhui University of Science and Technology. Athymic BALB/c nu/nu mice (4e6 weeks old) were fed under pathogen-free conditions and injected subcutaneously with 1  107 SK-hep1 cells

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Fig. 4. Art combined with Sor induces apoptosis of lung cancer corelated to mTOR and ERK inhibition in vivoIsolated tumors from mice models with treatments as described in Fig. 1 were sectioned for immunofluorescence. (Ae D) Tumor slides were co-stained with phospho-mTOR, cleaved-PARP and DAPI.(A) Representative images showing p-mTOR and c-PARP immunostaining.The percentage of pmTORþ (B) and c-PARPþ (C) tumor cells in isolated lung cancer samples from mice model. (D) The correlation between the percentages of p-mTORþ and c-PARPþ tumor cells in isolated lung cancer samples. (EeH) Tumor slides were co-stained with phospho-ERK, cleaved- PARP and DAPI. (E) Representative images showing p -ERK and c-PARP immunostaining. The percentage of p-ERKþ (F) and c-PARPþ (G) tumor cells in isolated lung cancer samples from mice model. (H) The correlation between the percentages of p-ERKþ and c-PARPþ tumor cells in isolated lung cancer samples. Mean ± SEM, **p < 0.01; ***p < 0.001; ****p < 0.0001 by Welch’s t-test, n ¼ 18 per group.

suspended in 100 ml PBS through 22-gauge needles. When tumor size reached approximately 50 mm3, mice were injected intraperitoneally with DMSO as control, or Sor (2.5 mg/kg) with or without Art (100 mg/kg), or Art alone once every other day for 4 weeks. Tumor sizes were measured with caliper in period. On the second day after the last administration, mice were sacrificed and tumors were isolated and weighted. For treatments, Sor and Art were resolved with DMSO (dimethyl sulfoxide) and diluted to working concentrations.

data were analyzed by FlowJo software. 4.4. MTS According to 5  103 cells per well, SM or SK cells were seeded in 96-well plates and treated with Sor, Art or DMSO as control for 24 h. Then, 20 ml MTS solution were pipetted into each well to incubate at 37  C for 2 h in a humidified incubator with 5% CO2, according to the kit instructions (Promega, cat #G3580).

4.2. Cell cultures 4.5. Western blot SK-hep1 and SM-7721 cell lines are characterized with low and high differentiation of HCC, respectively. Both cell lines were cultured with complete DMEM medium containing 10% heatinactivated fetal bovine serum and 100 U/ml penicillin/streptomycin and incubated at 37  C in a humidified incubator containing 5% CO2 [6]. 4.3. Flow cytometry Cells were suspended in 100 ml binding buffer and incubated with FICT-annexin V and PI for 25 min at cold ice in the dark, according to the kit instructions (BD, cat #556547). Then, labeled cells were analyzed using CANTO II flow cytometer (BD Biosciences) and

Cells were lysed in protein extraction reagent containing proteinase inhibitors on ice for 30 min. Equal cell lysis was transferred from SDS-polyacrylamide gels into PVDF membranes prior to labeling with primary antibodies at 4  C overnight and horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature [6,18e20]. Antibodies used including anti-caspase3 (Abcam, cat #ab32351), anti-phospho-S2448-mTOR (Abcam, cat #ab109268), anti-cleaved PARP (CST, cat #5625), anti-ACTB (PTG, cat #10230-1-AP), anti-GAPDH (PTG, cat #10494-1-AP), antiphospho-S338-cRAF (CST, cat #9427), anti-phospho-T202/Y204ERK1/2 (CST, cat #4370) and anti-phospho-S473-Akt (CST, cat #4060).

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4.6. Immunofluorescence Paraffin-embedded tumor tissue was cut into 4 mm slides for incubation with anti-cleaved PARP (Abcam, cat #ab110315), antiphospho-Y391-mTOR (Abcam, cat #ab32028), anti-phospho-T202/ Y204-ERK1/2 (CST, cat #4370), following labeled by FITCconjugated goat anti-rabbit IgG (PTG, cat #SA00003-2) or Rhodamine-conjugated goat anti-mouse IgG (PTG, cat #SA000071). 4.7. Statistics All statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). The relationship between markers was assessed by Pearson’s correlation and regression. For comparisons, two-tailed Student t-test and Welch’s t-test were applied, as appropriate. All values are mean ± SEM and P value < 0.05 was considered significant.

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Conflicts of interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.

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Acknowledgements

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This study was supported by the National Natural Science Foundation of China (No. 81971483, 81672445, 81571528). The funders had no role in the study design, data collection, analysis, and decision to publish or preparation of the manuscript.

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