Curcumin exerts anti-tumor effects on diffuse large B cell lymphoma via regulating PPARγ expression

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Curcumin exerts anti-tumor effects on diffuse large B cell lymphoma via regulating PPARg expression Wei Zhang a, b, 1, Qiong Li a, b, 1, Chao Yang a, b, Huan Yang a, b, Jun Rao a, b, *, Xi Zhang a, b, ** a b

Medical Center of Hematology, Xinqiao Hospital, Army Medical University, Chongqing, 400037, China State Key Laboratory of Trauma, Burns and Combined Injury, Army Medical University, Chongqing, 400037, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2019 Accepted 28 December 2019 Available online xxx

Given the highly heterogeneity of diffuse large B cell lymphoma (DLBCL) and the diverse demands for proper treatment, many patients would relapse or show resistance to current therapeutic regimens, new treatment options are urgent to be explored. Curcumin harbored anti-tumor potential in various cancers, here, we investigated the possible effects and mechanism of curcumin on human DLBCL in vitro and in vivo, we found that curcumin inhibited cell viability in a concentration and time dependent manner, promoted cell apoptosis and arrested cell cycle at G2 phase, and these effects were mediated by PPARg promotion and Akt/mTOR pathway inactivation. Furthermore, effects of curcumin on human DLBCL cells could be partly rescued by PPARg antagonist GW9662, and enhanced by PPARg agonist rosiglitazone. Taken together, our results demonstrated that curcumin inhibited the proliferation of DLBCL cells by upregulating the expression of PPARg, and our results might provide novel therapeutic approaches and a potential target to DLBCL treatment. © 2020 Elsevier Inc. All rights reserved.

Keywords: Curcumin Diffuse large B cell lymphoma PPARg Akt signaling pathway

1. Introduction Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma (NHL) and accounts for up to 35e40% of all cases. Based on the differentiation stage of B cells that they originated from, it can be divided into germinal center B-cell like and activated B-cell like DLBCL [1,2]. Despite the ReCHOP based chemotherapy regimen could improve the 5-year overall survival of DLBCL patients to approximately 60% [3], 30% patients would become refractory or relapse [4,5]. Therefore, it is of great clinical significance to find other effective therapeutic targets and strategies. Curcumin is a kind of plant polyphenol extracted from turmeric, which possesses anti-inflammatory and anti-oxidant effects [6]. Accumulating evidences indicated that curcumin exerted anticancer effects in various solid tumors by inducing apoptosis and inhibiting angiogenesis [7,8]. As for hematological malignancies,

in vitro studies showed curcumin could promote cell apoptosis by suppressing NF-kB signaling pathway in Burkitt’s lymphoma [9] and cutaneous T-cell lymphoma [10]. Other in vivo studies demonstrated that curcumin could inhibit the growth of Hodgkin’s lymphoma xenografts [11], decrease the secretion of interleukin-1 (IL-1a and IL-1b) [12], tumor necrosis factor-a and interleukin-6 in T-cell lymphoma bearing mice [13]. All these results demonstrated that curcumin possessed an anti-lymphoma capacity, but whether it harbors inhibitory effect on DLBCL is not clear, and the underlying mechanism needs to be explored. In present study, we investigated the effects of curcumin on apoptosis, proliferation and chemosensitivity of DLBCL cell lines with different concentrations. We found that PPARg was a critical factor inducing the effects of curcumin, this study demonstrated the inhibitory effect of curcumin on DLBCL in vitro and in vivo, and further explored its possible mechanism, and our results might provide experimental evidence for curcumin clinical application. 2. Materials and methods

* Corresponding author. Medical center of hematology, Xinqiao Hospital, Army Medical University, Chongqing, 400037, China. ** Corresponding author. Medical center of hematology, Xinqiao Hospital, Army Medical University, Chongqing, 400037, China. E-mail addresses: [email protected] (J. Rao), [email protected] (X. Zhang). 1 These authors contributed equally to this study.

2.1. Reagents Human DLBCL GCB subtype cell line Ly1 and ABC subtype cell line Ly3 were obtained from Beckman Research Center (City of

https://doi.org/10.1016/j.bbrc.2019.12.129 0006-291X/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: W. Zhang et al., Curcumin exerts anti-tumor effects on diffuse large B cell lymphoma via regulating PPARg expression, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.129

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hope, USA). IMDM medium, fetal bovine serum (FBS) were purchased from Hyclone (UT, USA). Curcumin was purchased from Sigma (MO, USA), CCK8 kit was purchased from DOJINDO (Shanghai, China), the PE Annexin V Apoptosis Detection kit was purchased from BD (CA, USA), the antagonist GW9662, BCA kit were purchased from Beyotime biotechnology (Shanghai, China), the agonist Rosiglitazone was purchased from MCE (NJ, USA), Rabbit anti-human p-Akt, p-mTOR, Akt, mTOR, b-actin antibodies were purchased from CST(MA, USA), and Rabbit anti-human PPARg antibody was purchased from Abcam (MA, UK). The CHOP regimen: cyclophosphamide, hydroxydoxorubicin, oncovin and prednisone was purchased from Sigma (MO, USA). 2.2. Cell cultures Cells were cultured in IMDM medium containing 10% FBS, and maintained at 37
C in a 5% CO2 incubator.

formula: (0.5  length  width2). Then tumor tissues were dissected and fixed in 10% formalin preparing for H&E staining and IHC staining. The animal experiments were approved by the Institutional Animal Care and Use Committee of the Xinqiao Hospital, AMU and strictly complied with the recommendations in the Guide for the Care and Use of Laboratory Animals of AMU. 2.7. Immunohistochemical (IHC) staining After fixed with 10% formalin and paraffin-embedded, the mice tissues were cut into 4-mm-thick sections and then deparaffinized with xylene, hydrated with series diluted alcohol, blocked the endogenous peroxidase activity with hydrogenperoxide and then repaired antigen with sodium citrate solution according to standard procedures. Subsequently, sections were incubated with PPARg and Ki-67 antibodies at 4
C overnight, after washing with PBS, incubated with the secondary antibody at room temperature for 30min. At last, washed sections were stained with BAD.

2.3. Cell viability evaluation 2.8. Statistical analysis Cell viability was detected by CCK8 kit, groups were different according to each experiment aim with at least 3 duplicate wells. Once grew to the logarithmic phase, cells were resuspended and incubated into 96-well plates in an amount of 20000 cells per well. When cell viability was evaluated, each well added with 10ul CCK8 solution and incubated in the incubator for 2e4 h before measuring the absorbance at a wavelength of 450 nm. 2.4. Flow cytometry analysis for cell apoptosis and cell cycle After treatment, cells were harvested and washed with PBS for 2e3 times. When detecting the cell apoptosis, cells were incubated with Annexin V and 7-AAD for 15 min at 37
C in the dark according to the instructions; when detecting the cell cycle, the washed cells were fixed with precooled 70% absolute ethanol at 4
C overnight, and then stained with propidium iodide supplemented with RNase A. Then cell apoptosis and cell cycle were detected with BECKMAN Flow cytometry. 2.5. Western blot The total protein was extracted and concentrations were assessed by BCA kit, proteins were separated via 12% SDS-PAGE gel and transferred to a PVDF membrane, then blocked the membrane with TBST containing 5% defatted milk on a shaker at room temperature for 2 h, after that incubated with corresponding primary antibodies at 4
C overnight, washed with TBST before incubated with horseradish peroxidase (HRP) -labeled secondary antibody on a shaker at room temperature for 2 h, then washed as above, finally detecting protein expression using enhanced chemiluminescence (ECL) kit. 2.6. Xenograft mouse model Six-week NOD/SCID mice (SPF Biotechnology Co.,Ltd. Beijing, China) were housed under IVC environment with controlled conditions. Xenograft tumors were generated by subcutaneous injection of 5  106 Ly1 cells dispersed in 0.2 mL PBS: Matrigel (1:1). When tumor volume reached to approximately 100 mm3, mice were randomly divided into three groups (n ¼ 5): (a) control group: PBS; (b) curcumin group: 200 mg/kg; (c) curcumin and rosiglitazone group: curcumin (200 mg/kg) þ rosiglitazone (50 mg/kg). All treatments were dispersed in 0.1 mL PBS and injected intraperitoneally every other day for 14 days. Tumor size was measured before injection every 4 days and the values were calculated using the

All experiments were repeated at least three times. Data were depicted as the mean ± SE. The software used for statistical analysis was SPSS 25.0. Comparisons between groups were performed by ttest. p < 0.05 was considered as a statistically significant difference. 3. Results 3.1. Curcumin inhibited the cell viability of DLBCL in a concentration and time dependent manner in vitro To investigate the effect of curcumin on cell viability of DLBCL cells, IC50 of Ly1 and Ly3 cells were evaluated after treated with curcumin for 12 h, the results showed that IC50 of Ly1 was 38.98 mM, and Ly3 was 30.68 mM (Fig. 1AeB), then we cultured cells in the presence of different concentrations of curcumin (0, 5, 10, 15, 20, 40 mM) for 12 h and 24 h, respectively, CCK8 assay was used to detect the survival rates, cell viability markedly decreased in both cell lines in a concentration-dependent manner, and the inhibition effect after treated for 24 h was much more remarkable than that of 12 h (Fig. 1CeD). Then, 5 mM and 10 mM curcumin were selected for different treatment times (0, 12, 24, 48, 72 h), the cell viability decreased in a time-dependent manner in both cell lines (Fig. 1EeF). Taken together, these results showed that curcumin could inhibit DLBCL cell proliferation in a concentration and time dependent manner. 3.2. Curcumin promoted DLBCL cells apoptosis and arrested cells at G2 phase 5 mM and 10 mM curcumin were selected as the working concentrations for following experiments, after incubated with curcumin for 24 h, Ly1 and Ly3 cells were harvested to detect the apoptosis and cell cycle by flow cytometry. The apoptosis rate of 10 mM curcumin group was significantly higher than that of 5 mM curcumin group and control group in Ly1 cells (8.33 ± 0.52%, 4.89 ± 0.24%, 2.61 ± 0.20%, P < 0.05) and Ly3 cells (12.02 ± 0.21%, 6.63 ± 0.21%, 2.62 ± 0.15%, P < 0.05) (Fig. 2A, C). Cell cycle alterations were also evaluated, compared with control, the proportion of G2 phase cells of 5 mM curcumin and 10 mM curcumin group were significantly increased in Ly1 cells (5.94 ± 0.25%, 13.74 ± 0.40%, 25.37 ± 0.20%) and Ly3 cells (10.90 ± 0.38%, 19.88 ± 0.24%, 27.16 ± 0.57%) (Fig. 2B, D). Moreover, cell chemosensitivity to CHOP regimen was investigated, curcumin could decrease the IC50 of cells to CHOP chemotherapy regimen from 332.8 ng/ml to

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Fig. 1. Effects of curcumin on cell viability of DLBCL. CCK8 assay was used to detect IC50 of Ly1 (A) and Ly3 (B) after treated for 12 h, cell viability of Ly1 (C) and Ly3 (D) after treated for 12 h and 24 h, and cell viability of Ly1 (E) and Ly3 (F) after treated with 5 mM and 10 mM curcumin for 0, 12, 24, 48, 72 h. Data are presented as mean ± SE of three independent experiments.

202.1 ng/ml in Ly1, and from 406.2 ng/ml to 289.9 ng/ml in Ly3 (Fig. 2E and F). 3.3. Curcumin inhibited Akt/mTOR signaling pathway via upregulating PPARg expression In order to figure out the underlying mechanism of the curcumin anti-lymphoma effect, potential targets of curcumin were searched through DrugBank database (https://www.drugbank.ca/ drugs/DB11672), and we found peroxisome proliferator-activated receptor gamma (PPARg) was one of the potential curcumin targets. Then, we examined the expression of PPARg after treatment with curcumin, our results showed that 10 mM curcumin treatment could up-regulate PPARg expression with a time-dependent manner in both cell lines. Besides, as PI3k/Akt/mTOR pathway is well known for cell survival and apoptosis regulation, we evaluated the involvement of this signaling pathway at the same time, and found that p-Akt and p-mTOR proteins gradually decreased, while cleaved caspase3 protein increased (Fig. 3A). Furthermore, in order to explore whether the inactivation of PI3k/Akt/mTOR pathway was mediated by PPARg, both cells lines were incubated with 10 mM PPARg antagonist GW9662 and 20 mM agonist rosiglitazone for 24 h, respectively. Results showed that after incubated with rosiglitazone, the p-Akt and p-mTOR protein decreased, the cleaved caspase3 protein increased, but after incubated with GW9662, increasing p-Akt and p-mTOR and decreasing cleaved caspase3 were observed (Fig. 3B). Overall, these results demonstrated PI3k/Akt/mTOR pathway was downstream of PPARg, and curcumin might exert anti-lymphoma effect through upregulation PPARg. 3.4. PPARg antagonist could reverse the effects of curcumin on DLBCL cells To further investigate the role of PPARg in the effects of curcumin

on DLBCL, Ly1 cells were incubated with 10 mM curcumin combination with GW9662 or rosiglitazone for 24 h, cells were harvested to detect the apoptosis and cell cycle by flow cytometry. Compared with the curcumin group, the apoptosis rate of curcumin þ GW9662 group decreased, while curcumin þ rosiglitazone group increased (6.13 ± 0.14%, 5.60 ± 0.03%, 6.52 ± 0.14%) (Fig. 3C, E). Meanwhile, the proportion of G2 phase cells of curcumin þ GW9662 group decreased, while curcumin þ rosiglitazone group increased (11.41 ± 0.47%, 8.77 ± 0.31%, 14.91 ± 0.30%) (Fig. 3D and E). Taken together, the promotion of apoptosis and arrestment of G2 phase mediated by curcumin could be partly reversed by PPARg antagonist, and enhanced by PPARg agonist. Next, we examined the effects of PPARg on the inactivation of Akt pathway mediated by curcumin, Ly1 cells were incubated with 10 mM curcumin combination with different concentrations of GW9662 (10, 20 mM) or rosiglitazone (20, 40 mM) for 24 h. Western blot results showed that compared with the curcumin group, p-Akt, p-mTOR proteins increased in curcumin þ GW9662 group, on the contrary, the inhibition of Akt pathway was strengthened in curcumin þ rosiglitazone group (Fig. 3F). All these results demonstrated that inactivation of Akt pathway mediated by curcumin could be partly reversed by PPARg antagonist, and partly enhanced by PPARg agonist.

3.5. Curcumin inhibited DLBCL growth in vivo We established a Ly1-transplanted NOD/SCID mouse model to evaluate the effect of curcumin on DLBCL in vivo. Mice were randomly divided into three groups (n ¼ 5) and subcutaneously injected with PBS, curcumin, curcumin and rosiglitazone, respectively. Consistent with our results in vitro, curcumin showed a DLBCL growth inhibition ability, furthermore, rosiglitazone enhanced the growth inhibition induced by curcumin (Fig. 4A and B). Then tumor tissues dissected from curcumin and PBS injected mice were used for Ki-67 and PPARg staining, results demonstrated

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Fig. 2. Effects of curcumin on DLBCL cell apoptosis, cell cycle and cell chemosensitivity to CHOP. Flow cytometry was used to detect the apoptosis and cell cycle of Ly1 and Ly3 after treated with 5 mM and 10 mM curcumin for 24 h. Representative images of flow cytometric analysis for cell apoptosis (A) and cell cycle (B). Statistical results of flow cytometric analysis for cell apoptosis (C) and cell cycle (D). IC50 of Ly1 (E) and Ly3 (F) to CHOP chemotherapy regimen were examined through CCK8 assay. Data are presented as mean ± SE of three independent experiments. **p < 0.05.

that PPARg in tumor tissue with curcumin treatment was higher than that of control. (Fig. 4C). Taken together, curcumin could inhibit the proliferation of DLBCL via up-regulating PPARg expression in vivo.

4. Discussion Despite that ReCHOP was regarded as the standard therapy for DLBCL patients, up to 30% patients would experience relapse or refractory diseases following initial therapy [4,14], no standard regimen can be proposed and more novel agents need to be explored for these patients. Recently, several targeted drugs, such as the BTK inhibitor ibrutinib [15], the protease inhibitor bortezomib [16] had been applied in clinical trials and shown a promise response on patients, however, the long-term survival benefit was not clear. In our study, we found that curcumin could inhibit DLBCL cells proliferation, promote cells apoptosis and improve cell chemosensitivity to CHOP regimen, all these effects were mediated by upregulating PPARg expression. Our results provide a novel insight for DLBCL treatment and new anti-lymphoma strategies development. Curcumin has been proved to be a promising anticancer drug in alone or in combination with other drugs. The anticancer effects involved a variety of signaling pathways, for instance, the Wnt/bcatenin pathway, the JAK/STAT pathway and the NF-kB pathway

[17]. Meantime, several clinical trials of curcumin are still ongoing. In a randomized, double-blind, placebo-controlled trial of prostate cancer, curcumin significantly suppressed the PSA level and showed a better tolerance and safety in patients [18]. In a phase II clinical trial of advanced pancreatic cancer, combination of gemcitabin and Meriva (a phytosome complex of curcumin) showed an improved safety and efficacy of gemcitabine [19]. But in another double-blinded randomized study, cruz-Correa et al. found that there was no difference in the mean number or size of lower intestinal tract adenomas between patients given curcumin and those given placebo for 12 weeks, which might due to low dose of curcumin [20]. These indicated that combination of curcumin and chemotherapy drugs showed promising clinical outcome, but effect of curcumin applied alone might be limited by its purity and bioavailability. DrugBank database and other researchers suggested Peroxisome proliferators activated receptor g (PPARg) was the downstream target of curcumin [21,22]. PPARg is a member of type II nuclear hormone receptor superfamily, which can participate in the regulation of inflammatory response, cell proliferation and differentiation, it had also been reported to be expressed in various tumors, such as bladder cancer, breast cancer, thyroid cancer [23]. Notably, the function of PPARg on cancers was still the subject of debate, in bladder cancer, Cheng revealed that PPARg was upregulated in human bladder cancer tissues, and knockdown

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Fig. 3. PPARg mediated the effects of curcumin on DLBCL. Western blot was used to detect the PPARg, p-Akt, Akt, p-mTOR, mTOR, cleaved caspase3 expression in Ly1 and Ly3 after treated with 10 mM curcumin for different hours (0, 2, 4, 8,12 h) (A) and treated with 10 mM PPARg antagonist GW9662 and 20 mM agonist rosiglitazone for 24 h (B). Flow cytometry was used to detect the apoptosis and cell cycle of Ly1 after treated with 10 mM curcumin combine with 10 mM antagonist GW9662 or 20 mM agonist rosiglitazone for 24 h. Representative images of flow cytometric analysis for cell apoptosis (C) and cell cycle (D). Statistical results of flow cytometric analysis (E). Western blot (F) was used to detect the pAkt, Akt, p-mTOR, mTOR, cleaved caspase3 protein expressions in Ly1 after treated with 10 mM curcumin in combination with different concentrations of GW9662 (10, 20 mM) and different concentrations of rosiglitazone (20, 40 mM) for 24 h. Data are presented as mean ± SE of three independent experiments. **p < 0.05.

PPARg could significantly suppress cancer cells proliferation [24]. While other study showed that PPARg was a favorable prognostic in bladder cancer patients, PPARg activation resulted in inhibition of cell proliferation in vitro and suppression of tumor growth in vivo [25]. In present study, we found that curcumin could upregulate the expression of PPARg, and PPARg exertedanti-lymphoma effect by inactivation of PI3k/AKt signaling pathway (Fig. 4D). Furthermore,

PPARg antagonist GW9662 could reverse the G2 phase arrestment and proliferation inhibition induced by curcumin, and inactivation of Akt pathway induced by curcumin could be abrogated simultaneously, yet PPARg agonist rosiglitazone enable an enhanced effectiveness of curcumin. In conclusion, our results suggested that curcumin could inhibit DLBCL proliferation, promote cells apoptosis, arrest cells at G2 phase and improve the chemosensitivity via PPARg-mediated pathway.

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Fig. 4. Effects of curcumin on DLBCL growth in vivo. Ly1-transplanted NOD/SCID mice were randomly divided into three groups (n ¼ 5), and from day 24 after transplanted, three groups were intraperitoneally injected with PBS, curcumin, curcumin and rosiglitazone respectively every other day for additional 14d, tumor tissues were dissected on day 40 (A). Tumor size was measured before injection every 4 days (B), **P < 0.05. Representative H&E staining and IHC staining of Ki-67 and PPARg of tumor tissues dissected from curcumin and PBS injected mice (C). Schematic representation of curcumin mediated inhibition of DLBCL via upregulating PPARg expression and further inhibiting Akt signaling pathway (D).

Our finding revealed a novel target and offered a new strategy for the treatment of diffuse large B lymphoma. As PPARg was reported to be involved in reprogramming energy metabolism of tumors [26], whether curcumin could affect DLBCL glucose metabolism and the exact effects on different metabolic phenotype of DLBCL deserved to be further explored.

(2019XLC3020). We thank Dr. Jia Liu for their assistance with the statistical analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.129.

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Acknowledgements Grant support This project was supported by grants from the National Natural Science Fund for Youth (No. 81600166), the National Natural Science Foundation of China (No.81570097), the technique innovation and applied program of Chongqing (cstc2018jscx-msybX0052) and Science and technology innovation promotion project of AMU

Supported by grants from the National Natural Science Fund for Youth (No. 81600166), the National Natural Science Foundation of China (No.81570097), the technique innovation and applied program of Chongqing (cstc2018jscx-msybX0052) and Science and

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Please cite this article as: W. Zhang et al., Curcumin exerts anti-tumor effects on diffuse large B cell lymphoma via regulating PPARg expression, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.129