Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

Abstract Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.


| INTRODUC TI ON
and it is regulated by AKT. In normal cells, mTORC1 is essential for erythroid and megakaryocytic differentiation through the activation of downstream effectors including 4eBP1 and p70s6K. 6 This pathway has been found deregulated particularly in megakaryocytes of MPNs patients. 7 The deregulation of JAK/STAT and mTOR pathways induces an inflammatory state with aberrant cytokine expression. 8 Given the heterogeneous clinical needs of MPNs patients, determination of a standard therapeutic protocol is often difficult. Moreover, targeted therapy with JAK inhibitors revealed to have some limits in terms of efficacy, 9 therefore it is necessary to find additional approaches to improve the results so far obtained.
Curcumin is the active phytochemical component isolated from the rhizome of the Curcuma longa plant. Curcumin is a highly pleiotropic molecule with multiple pharmacological effects, such as antiinflammatory, anti-microbial, anti-oxidative and anti-proliferative activities. 10,11 Extensive preclinical trials have indicated curcumin therapeutic potential against a wide range of human diseases. 12 Previous studies showed that curcumin can suppress JAK2/STAT signalling pathways in different type of cancer and injuries. 13,14 Chen et al demonstrated that curcumin increased the transcript levels of SOCS-3, an important negative regulator of JAK2, and significantly inhibited the clonogenic activity of hematopoietic progenitors from MPNs patients. 15 Furthermore, curcumin was able to dissociate Raptor from mTOR, by inhibiting mTORC1 signalling and the phosphorylation of its downstream effectors in different cell lines. 16 In this study, we investigated the effect of curcumin on JAK2 V617F cell line and in primary cells from MPNs patients. Our results suggest that curcumin inhibits proliferation and activates cell death program by modulating JAK2/STAT and mTORC1 pathways.

| Patients cohort
After informed consent, human peripheral blood (PB) leucocytes were isolated by Buffy Coat procedure from 30 MPNs patients (24 were PV, 4 ET and 2 PMF; the median age was 63 years (range 20-86); 18 were males and 12 females) and 10 healthy donors. All samples obtained from patients were JAK2 V617F mutated. The study was approved by the ethic committee on 16 December 2015 (number of approval 212/2015).

| Protein extraction and immunoblotting
To perform immunoblotting analysis, we used human HEL cells and cells from MPNs patients. To isolate total protein content, samples were   Table 1  Quantification was performed using Image Lab program (Bio-Rad). Cruz Biotechnology). mTOR antibody (Table 1) for co-immunoprecipitation was added to the lysate (1 mg/mL) and incubated ON at 4°C. Protein A/G (20 μL) were added to the antibody and lysate mixture and incubated 1 hour at 4°C in a rotator. The mock control (beads and whole cell lysates without adding antibody) was used to exclude the false interaction of lysate proteins with the beads.

| Co-immunoprecipitation
Immunoprecipitates and mock controls were washed once with CHAPS buffer lacking NaCl and three times with CHAPS buffer containing 150 mmol/L NaCl. Samples were eluted in 5× Laemmli buffer at 95°C for 10 minutes and resolved on 6% SDS-PAGE as described above. mTOR-Raptor complex was evaluated using mTOR and Raptor antibodies (Table 1) as previously described.

| RNA extraction and qRT-PCR analysis
Total RNA was extracted using TRIzol Reagent (Ambion, Thermo

| Statistical analysis
Statistical analyses were performed using the two-tailed Mann-Whitney U test and paired t test. All the analysis with confidence level major of 95% are indicated like significant and marked as followed: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

| Curcumin induces apoptosis and inhibits viability in HEL cells
To explore the effects of curcumin on cell death and viability, HEL cells were treated with various concentrations of curcumin (0-30 µmol/L) for 24/48 hours, proliferation and apoptosis were evaluated by FACS and immunoblotting. The viability of the HEL cells exposed to curcumin was significantly lower compared to control cells even at low concentrations. In addition, the curcumin growth inhibitory effect was dose and time-dependent, reaching the maximum effect at the dose of 20 µmol/L for 48 hours, by reducing the proliferation of 93% ( Figure 1A). The results obtained evaluating apoptosis with Annexin V integrate and confirm viability analysis. In particular, we showed that the percentage of apoptotic cells increase of 2.1, 3.6, 5.0 and 5.9 times at 24 hours and 2.1, 4.7, 6.3 and 8.6 at 48 hours, respectively after treatment with 10, 15, 20, and 30 µmol/L of curcumin ( Figure 1B).
Furthermore, to investigate the intrinsic apoptosis at the protein level, treated HEL cells were harvested at 24 hours and analysed by Western blot. As shown in Figure 1C, the protein expression of cleaved caspase-3 was markedly increased in the curcumin-treated HEL cells according to drug concentration. In particular, a curcumin concentration of 30 µmol/L showed a sharp increase of cleaved caspase-3. This data confirmed the FACS analysis and indicated that curcumin induced apoptosis in a dose-dependent manner in JAK2 V617F-mutated cell line.

| Curcumin affects JAK2/STAT pathway in HEL cell line
To Indeed, we observed that phosphorylation of STAT5 was 60% reduced, while STAT3 was extremely sensitive to curcumin-mediated JAK2 inhibition and its phosphorylation decreased to 20% even at the lower curcumin dose and was completely blocked at higher

| Curcumin modulates mTORC1 signalling by the inhibition of PDK and AKT in HEL cells line
To investigate how curcumin affects mTORC1 pathway, HEL cells  5). B, Effect of curcumin on apoptosis (n = 5). C, Caspase-3 Cleaved expression was evaluated by SDS-PAGE. The GAPDH level was used as loading control. The relative intensity of each band is shown under the blot as fold change (FC) compared to untreated control, to which a value of 1 unit was assigned. Statistical analyses were performed using the two-tailed Mann-Whitney U test, comparing conditions two by two respect to not treated condition. All the analysis with confidence level major of 95% are indicated like significant and marked as followed: *P ≤ 0.05; **P ≤ 0.01.
for 24 hours and analysed by immunoblotting. Our results indicated that curcumin affects the principal modulator of mTORC1: AKT.
Phosphorylation of AKT in Thr308 was reduced in dose-dependent manner and its principal activator PDK was inhibited by low dose curcumin and it appeared unphosphorylated at the maximum concentration utilized (Figure 3). The consequence was the reduction of the complex between mTOR and Raptor, as observed in Figure 3, especially at the highest drug concentration. Furthermore, by analysing the signal cascade, we observed the deregulation of the principal downstream effectors of mTORC1. In particular, we showed the dephosphorylation of p70s6K, which is usually activated by phosphorylation, and the increase of unphosphorylated form of 4eBP1, usually inhibited through mTORC1 phosphorylation.

| Curcumin induces apoptosis and reduces PIM family members and CD177 expression in JAK2 mutated MPNs patients
Due to the promising results in vitro, we tried to evaluate the effect of curcumin also in patient specimens. For these purposes, leucocytes from JAK2 mutated patients were treated with curcumin at the high- As a consequence, we observed a strong inhibition of mTORC1 downstream effectors 4eBP1 and p70s6K phosphorylation, both involved in cell growth and proliferation. 16   . Statistical analyses were performed using paired t test to compare treated and not treated MPNs patients' cells and the two-tailed Mann-Whitney U test to compare treated patients with healthy donors' cells. All the analysis with confidence level major of 95% are indicated like significant and marked as followed: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. B, Caspase-3 Cleaved expression was evaluated by SDS-PAGE. The GAPDH level was used to normalize data. Representative Western blots of three patients and three healthy controls were shown In conclusion, this study showed that curcumin exerts an antitumor effect on human JAK2-mutated cells by inducing apoptosis and inhibition of proliferation, through the regulation of both JAK2/STAT and mTORC1 pathways. These findings suggest that curcumin seems to be a promising nutraceutical compound that should be further evaluated in different pharmaceutical formulation for the treatment of MPNs.

ACK N OWLED G EM ENTS
The study was funded by grants from AIRC. The study was partially funded by a special grant from 'AIRC 5 per mille' to the AGIMM group (AIRC-Gruppo Italiano Malattie Mileoproliferative); for a complete list of AGIMM investigators see at http://www.progettoagimm.it.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
JP and VR designed the study, performed the experiments and wrote the manuscript. ML analysed data and revised the manuscript.