The synergistic effect of PFK15 with metformin exerts anti-myeloma activity via PFKFB3
Xinling Liu a, Yi Zhao a, Enfan Zhang a, Haimeng Yan a, Ning Lv b, Zhen Cai a, *
a Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
b Department of Pharmacy, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

a r t i c l e i n f o

Article history:
Received 17 May 2019
Accepted 21 May 2019 Available online xxx

Keywords: Myeloma Metabolism PFKFB3 PFK15
a b s t r a c t

High glucose metabolism provides sufficient energy for cancer cells and is enabled by metabolic en- zymes. PFKFB3 (6-phosphofructo-2-kinase) accelerates the synthesis of fructose 2,6-bisphosphate (F2,6P2), which is a powerful allosteric regulatory activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme of glycolysis. The aim of this study was to investigate the anti-myeloma function and underlying mechanism of suppressing PFKFB3 via PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2- propen-1-one). The role of PFK15 in killing multiple myeloma (MM) cells was evaluated by cytotox- icity and apoptosis assays, flow cytometry and Western blotting. The oral hypoglycemic drug metformin (Met) was found to inhibit PFKFB3 protein expression by gene chip and Western blotting techniques in our study. PFK15 also demonstrated a synergistic effect with metformin to eliminate MM cells. Taken together, our findings indicate that PFK15 inhibits MM cell proliferation through the PFKFB3/MAPKs/ STAT signaling pathway. The combination therapy of PFK15 and metformin may be a promising anti- cancer drug regimen for the treatment of MM.

© 2019 Published by Elsevier Inc.

⦁ Introduction

Multiple myeloma (MM) is a malignant hematologic disease defined by abnormal proliferation of monoclonal plasma cells. The incidence rate of MM is second only to that of lymphoma among all hematological malignancies [1]. Individuals with MM have several major clinical symptoms, including increased blood calcium levels, renal insufficiency, anemia, and bone lesions (CRAB). Novel treat- ment options (bortezomib, lenalidomide, pomalidomide, histone deacetylase inhibitors, and others) have contributed to a doubling in the average life expectancy of MM patients [2,3]. However, at present, MM remains incurable, and drug resistance is a tough problem of great significance. Finding effective new drugs to treat MM still represents a challenge.
Many cancer cells overexpress glucose metabolism enzymes, which contributes to the Warburg effect [4]. As one of the rate- limiting glycolytic enzymes, PFKFB3(6-phosphofructo-2-kinase) plays a vital role in synthesizing F2,6P2 to activate PFK-1. Many

* Corresponding author. Bone Marrow Transplantation Center, the First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79, Qingchun Rd. Hangzhou, Zhejiang Province, 310006, China.
E-mail address: [email protected] (Z. Cai).
studies have demonstrated that PFKFB3 protein level are highly overexpressed and are poor diagnostic markers in solid cancers, including breast and gastric cancers and human head and neck squamous cell carcinoma [5e7]. In addition, multiple oncogenes have been reported to be linked with PFKFB3, such as AMP- activated protein kinase (AMPK) [8], phosphatase and tensin ho- molog (PTEN) [9], mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K/Akt) [10], and mammalian target of rapamycin (mTOR) [11].
PFK15 is a specific inhibitor of PFKFB3. It displays high anti- tumor functions in various cancers. To date, there are few reports about the function of PFK15 in MM. In this study, we aimed to determine whether PFK15 has antitumor activity and the possible mechanism in MM. The effect of PFK15 combined with hypogly- cemic drug metformin (Met) on MM was also investigated in this study. We believe that exploring the mechanism of PFKFB3 in MM will provide a new theoretical basis for clinical treatment.

⦁ Materials and methods

Human myeloma cell lines, drugs. The human MM cell lines RPMI8226, ARP-1 and OPM2 were provided by Dr. Qing Yi (Department of Cancer Biology, Lerner Research Institute, Cleveland 0006-291X/© 2019 Published by Elsevier Inc.

Clinic, OH, USA). PFK15, metformin and LY2228820 were all pur- chased from Selleck (Houston.TX,USA). Primary CD138 cells from bone marrow of MM patients and normal healthy donors’peripheral blood mononuclear cells (PBMC) as the control were obtained after approval by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine.
Glucose uptake and F2,6P2measurements. For glucose uptake measurements, we utilized a glucose uptake assay kit (colorimetric, Abcam) and followed the manufacturer’s instructions to operate. Intracellular F2,6P2 concentration was measured following the previously described method [12].
Cell proliferation assay and synergy analysis. Cell growth was assessed using trypan blue exclusion test (Sigma, USA). We seed 1 × 105 cells in 12-well plates with different drug treatments for 24, or 48 h. Then, 100 ml cells was added to 10 mltrypan blue and incubated at 37 ◦C for 2 h. Absorbance was measured at 450 nm using a microplate reader (Bio Rad, Model 680). CompuSyn soft- ware (Biosoft, Cambrige, UK) was used to verify the effect of
different combination treatment groups (PFK15, metformin or their combination) at 24 h. The synergy analysis required that PFK15 was mixed with metformin in a constant ratio at a dosage determined by the IC50 of each drug. The three combination effects (synergism, additive effect and antagonism) were judged by a combination index (CI), which were respectively CI < 1, CI 1 and CI > 1.
Apoptosis and cell cycle analysis by flow cytometry. Cells were collected and incubated with Annexin VeFITC/PI according to the manufacturer’s instructions. The apoptotic cells were detected us- ing flow cytometry, and the data were analyzed with FlowJo 7.6.1 software. To detect cell cycle changes, the cells were washed twice with PBS and then fixed with precooled 75% ethanol at 4 ◦C over- night. The next day, after washing twice with PBS and treating with 0.01% RNase A for 30 min at 37 ◦C, the cells were then incubated with 0.5% PI. Flow cytometry (BD Biosciences, CA, USA) and ModFit software (version 3.2, Verity Software House) were then used to detect cells and analyze the data results.
Reverse transcription-quantitative polymerase chain reaction (RT- qPCR) analysis. To analyze the expression levels of target genes, total RNA from MM cells was extracted using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed using SYBR Green PCR kits (Takara, Japan). The following primer sequen- ceswere used: human PFKFB3 forward, 50-CTGGACAGGGAGGGA- GATACTA-30 and reverse, 50-AATGAAGAGCTTTGCCCGTGGTC-30;
human GAPDH forward, 50-ACGGATTTGGTCGTATTGGGC-30 and
reverse, 50-TTGACGGTGCCATGGAATTG -30. GAPDH served as an endogenous control.
Western blot analysis. Cell pellets were extracted with 30 ml RIPA (Beyotime, P0013C). The metamorphic proteins (20e40 mg) were separated by 4e20% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Germany). The membranes were blocked with 5% nonfat milk for 1e2 h, incubated with specific primary antibodies overnight, washed using Tris-buffered saline with Tween 20 (TBS-T), and incubated with horseradish peroxidase (HRP)econjugated anti-rabbit or anti-mouse antibodies at room temperature for 1 h. The membranes were washed with TBS-T three times again, and the image was detected using the ChemiDoc MP Imaging System (Bio-Rad) with an enhanced chemiluminescence detection kit for HRP (Biological Industries, Israel, Beit Haemek Ltd.). The amount of the protein of interest, whichwas expressed using arbitrary densitometric units, wasnormalized to the densi- tometric units of GAPDH.
Lentiviral vectors construction and gene transfection. Lentiviral vectors were constructed by GenBank. The overexpression sequence PFKFB3 was designed and synthesized by Shanghai GenePharma Co. Ltd. MM cell lines were plated in 6-well culture
plates (1 105 cells per well) containing serum-free DMEM me- dium. The lentivirus was successively added into the medium ac- cording to the multiplicity of infection 10-40 and was fully mixed. After 12 h, the medium containing virus was replaced with fresh 10% serum medium. Then, we observed the cell growth status after 72 h with an inverted fluorescence microscope.
Statistical analysis. All results were reported as the means ± standard deviations (SD). A two-tailed Student’s t-test was applied to determine the significant differences between two groups, and a one-way analysis of variance was applied to estimate the differences among three or more groupswith post hoc contrasts by StudenteNewmaneKeuls test. All P values less than 0.05 were considered to be significant. All analyses were performed using GraphPad Prism 5.0 (GraphPad Software, CA, USA). All the experi- ments were repeated three times.

⦁ Results

PFK15 inhibits MM cell proliferationand suppresses glucose uptake and intracellular levels of F2,6P2. First, expression of PFKFB3 in MM cell lines was higher compared with healthy donors’ PBMC which was detected by RT-qPCR as shown in Fig. 1A. PBMC came from two different donors’peripheral blood mononuclear cells as the control. PFK15 is a highly active substance that inhibits PFKFB3, and its anti- proliferative effects on myeloma cells were dose- and time- dependent, as shown in Fig. 1B. The 50% inhibitory concentration (IC50) of PFK15 was 3 mM at 48 h. With 2 mM PFK15, the glucose uptake rate and F2,6P2 levels were reduced (Fig. 1C).
PFK15 induces apoptosis and cell cycle arrest in MM cells. To assess the apoptotic effect of PFK15, MM cell lines were incubated with different doses of PFK15 for 24 h. Apoptotic cells were identified by an Annexin V/PI staining assay. Annexin V-positive cells were apoptotic in Fig. 2A; PFK15 had a notably pro-apoptotic effect on RPMI8226 and OPM2 cell lines. As shown in Fig. 2B, the number of TUNEL-positive MM cells increased after treatment with various PFK15 concentrations for 24 h. To study the mechanism of cell cycle arrest after PFK15 inhibition, RPMI8226 and OPM2 cells were incubated with or without 2 mM PFK15 for 24 h. Flow cytometry was used to detect cell cycle arrest. As shown in Fig. 2C, with PFK15 treatment, the cell cycle was inhibited in the S phase compared with the vehicle group. Western blotting further verified the changes in the expression levels of apoptosis-related and cell cycle- related proteins. As shown in Fig. 2D, Caspase-3, Bak and PARP-1 were notably activated after treatment with PFK15 (0e3 mM). The expression of the anti-apoptotic protein Mcl-1 was decreased. The expression of cell cycle proteins, such as P21, was significantly increased, and Cyclin D1 was markedly downregulated.
PFK15 inhibits the proliferation of MM cell lines through the PFKFB3/MAPKs/STAT signaling pathway. Previous studies have revealed that PFKFB3 is linked with the MAPK family (Erk, JNK, and P38) in cancer cells [10,13]. The MM cell lines RPMI8226 and OPM2 were treated with various concentrations of PFK15 for 24 h. Our results indicated that PFK15 decreased the phosphorylation of MAPK family members (Erk, JNK, and P38) and their downstream molecular target STAT1, as shown in Fig. 3A. To verify this signaling pathway, PFKFB3 was overexpressed in RPMI8226 and OPM2 cell lines (Fig. 3B, upper panel), and the MAPK protein expression level increased; meanwhile, additional PFK15 decreased the signaling pathway in both of the cell lines, as shown in Fig. 3B. Then, we added LY2228820 to change P38 MAPK protein expression, and the protein level of PFKFB3 remained unchanged, which demonstrated that PFKFB3 was located upstream in signaling pathway as shown in Fig. 3C. Moreover, LY2228820 is a P38 inhibitor, the mechanism of activation of p-P38 when added LY2228820 is not yet clear. Campbell RM et al. [14] demonstrated that LY2228820 potently and

Fig. 1. Effect of PFK15 on myeloma cell viabilityand the intracellular levels of F2,6P2 in MM cells. A, Expression of PFKFB3 in MM cell lines was detected by RT-qPCR; PBMC: normal healthy donors’peripheral blood mononuclear cells as the control. B, Cell viability was evaluated by trypan blue exclusion test in cell lines at 24 and 48 h after culturing in different doses of PFK15. C, Glucose uptake and F2,6P2 levels were measured. The data were obtained from three independent experiments and are presented as the mean ± SD. *P < 0.05.

selectively inhibited P38a MAPK substrate phosphorylation (p- Thr334-MK2) with no effects on phosphorylation of p38a MAPK and it mainly reduced downstream p38 MAPK signaling [15], therefore we speculated that upregulation of p-P38 by LY2228820 may be due to a negative feedback response.
PFK15 displays synergistic anti-myeloma activity with metformin. The results of gene chip in our group demonstrated that metformin decreased PFKFB3 level (data not shown). We determined PFKFB3 mRNA levels in MM patients (n 12)and patients with MM and diabetes at the same time (here in referred to as MD patients) (n 7). As shown in Fig. 4A, PFKFB3 mRNA expression displayed higher levels in MD patients (left panel) and metformin signifi- cantly decreased PFKFB3 protein expression in MM cell lines (right panel). Because PFK15 could inhibit glucose uptake and cause anticancer cell activity, we further examined the relationship (synergistic, additive or antagonistic effect) between PFK15 and metformin with CompuSyn software, RPMI8226 and OPM2 cell lines were treated with PFK15 alone, metformin alone or their combination. As shown in Fig. 4B, the IC50 of metformin was 10 mM (upper panel), which is based on the results by Zi FM et al. [16]. According to the IC50 of metformin in RPMI 8226 and OPM2 cell lines (upper panel) and the IC50 of PFK15, MM cell lines were first treated with PFK (1, 2, 4 mM), with or without metformin (10, 20, 40 mM), or with their combination at a 1:10 ratio to test cell viability with trypan blue (middle panel) and PFK15 plus metfor- min showed a significant synergistic effect according to CI < 1
(lower panel). To further verify the synergistic mechanism of the combination, MM cells were treated with PFK15 with or without metformin, and the apoptotic cell proportion was detected by flow cytometry. As shown in Fig. 4C and D, compared with the group that only received PFK15, the percentage of apoptotic cells was dramatically increased in the combination treatment group.

⦁ Discussion

Multiple myeloma (MM) remains challenging despite the advent of novel drugs. Aberrant high glycolytic flux is a unique biological characteristic that was discovered in various cancer cells [17]. Discovering new glycolysis inhibitors may be promising for therapeutic advances [18].
Oncogenes and rate-limiting enzymes are involved in aberrant glycolysis [19e21]. One of the glycolytic enzymes in cancer cells is phosphofructokinase-2/fructose-2,6-bisphosphatase (PFKFB), which has 4 isoforms, PFKFB1 to PFKFB4. PFKFB3 has a relatively high kinase activity among the four isoforms. Although many pharmacological studies reported that inhibiting glycolysis (War- burg effect) is a potential anticancer approach, recent genetic studies, in contrast, demonstrated that Warburg effect is dispens- able for tumor growth [22,23]. Pouyssegur's lab however demon- strated that targeting lactic acid export (MCT1/MCT4) suppressed tumor growth [24]. This tumor growth arrest, in contrast to LDHA/ B-Double Knockout [22], was due to intracellular acidification, a

Fig. 2. Effect of PFK15 on myeloma cell apoptosis and cell cycle arrest. A, Results showing the apoptotic rate of RPMI8226 and OPM2 cells after treatment with or without PFK15 (0, 1.5, or 3 mM) for 24 h as detected by flow cytometry. Histograms showing the percentage of cells in apoptosis. Annexin V-positive cells were considered to be apoptotic cells.
*P < 0.05, **P < 0.01 (PFK15 vs vehicle). B, TUNEL assays indicated that PFK15 induced apoptotic cell death in MM cells (white arrow). All the experiments were repeated three times. C, Flow cytometry results displaying the percentage of cells in each phase of the cell cycle (G1, S or G2 phase) in RPMI8226 and OPM2 cells without or with 2 mM PFK15 treatment for 24 h. Histograms showing the proportion of G1, S and G2 phases of myeloma cells in three independent experiments. D, Changes in key proteins, such as cleaved Caspase-3, Bak, PARP-1, Mcl-1, P21 and Cyclin D1 after PFK15 treatment, as detected by Western blotting. All the experiments were repeated three times.

negative effector of mTORC1 and glycolysis [24]. And tumorigenesis based on genetic alterations was also shown to be influenced by PFKFB3 in many cancer cells. Moreover, Brian F. Clem et al. found a novel PFKFB3 inhibitor named PFK15 that markedly reduced the glucose metabolism and proliferation of cancer cells [25].
In this study, we demonstrated that PFK15 reduced glucose
uptake and F2,6P2 levels and had antitumor effects in MM cell lines in a dose- and time-dependent manner. Metformin, as an antidia- betic drug, has demonstrated its antitumor function. Meanwhile, we found metformin suppressed PFKFB3 expression, and MM pa- tients with diabetes had higher PFKFB3 protein expression. More- over, the inhibitory action on mitochondrial complex I of

Fig. 3. Mechanism studies of the anti-myeloma effect of PFK15. A, PFK15 repressed phosphorylated levels of MAPK (Erk JNK P38)/STAT signaling proteins in MM cell lines, as detected by Western blot. B, Overexpression of PFKFB3 in two cell lines increased the phosphorylation and expression levels of MAPK/STAT protein, and extra PFK15 counteracted some of the effects of PFKFB3overexpress. C, The P38 MAPK inhibitor LY2228820 did not change the protein level of PFKFB3, which suggested that PFKFB3 is located upstream in the signaling pathway. In Fig. 3B and C, the representative results of Western blotting were verified in RPMI8226 cell lines. All the experiments were repeated three times.

Fig. 4. Remarkable combined effect of PFK15 and metformin. A, MM patients and MD patients have high PFKFB3 mRNA levels. Metformin decreased PFKFB3 protein level; the representative result of Western blotting was verified in RPMI8226 cell line. B, The IC50 of metformin was 10 mM (upper panel), and from Figs. 1 and 2, IC50 of PFK15 was 3 mM. Cell viability was tested with PFK (1, 2, or 4 mM), with or without metformin (10, 20, or 40 mM) or their combination at a 1:10 ratio (medium panel). According to the IC50 of each drug, the Fa-CI plot curves also showed that PFK15 had a synergistic effect with metformin (CI < 1) using CompuSyn software (lower panel). C-D, Histograms showed the proportion of apoptotic RPMI8226 and OPM2 cells treated with PFK15 (2 mM), metformin (20 mM), or the combination for 24 h, as detected using flow cytometry. *P < 0.05, **P < 0.01.

metformin has already been found [26,27]. Based on above find- ings, we combined PFKFB3 inhibitor PFK15 with metformin together and discovered their synergistic effect in anti-myeloma activity. It is uncertain whether clinical treatment will be practi- cable by stopping glycolysis and OXPHOS. According to our exper- imental results, we propose that PFKFB3 inhibitor plus metformin combined with traditional chemotherapy drugs may improve therapeutic effect in MM patients with diabetes.
Then, we tried to understand the possible mechanisms of the anti-myeloma activity of PFK15. Using flow cytometry analysis, we found that PFK15 induced cell cycle arrest in S phase compared with control cells and accelerated MM cell apoptosis, as shown by Annexin V and PI staining. Western blot analysis further revealed that cleaved Caspase 3, Bax, P21, and PARP-1 were activated and Mcl-1 and CyclinD1 were repressed. While most of studies have shown that P21 and Cyclin D1 are the protein of G1 phase arrest, however, other studies indicated that P21 and Cyclin D1 were also involved with S phase arrest [28,29] which were consistent with our results.
Several major signaling pathways are closely related to prolif- eration, survival, migration and drug resistance of in MM cells, such as PI3K/Akt/mTOR, Ras/Raf/MEK/MAPK, JAK/STAT, NF-kB, Wnt/b- catenin, and RANK/RANKL/OPG [30]. The mitogen-activated protein kinase (MAPK) signaling pathway contains extracellular signal- related kinases (Erk), Jun amino-terminal kinases (JNK), and P38- MAPK. Previous studies have shown that suppressing the MAPK/ Erk signaling pathway might inhibit viability and induce apoptosis in cancer cells [31e33]. It has also been demonstrated that Erk is involved in nitric oxide-induced glycolysis [34]. JNK-related path- ways play vital roles in cell proliferation and cell death [35e38]. JNK also regulates metabolic functions in mitochondria to link cytosolic and mitochondrial processes [39]. P38 MAPK inhibition enhanced MM cell lines' apoptosis induced by different drugs and was effective in reducing tumor volume in a the mouse xenograft model of multiple myeloma [40]. However, the effect of the MAPK family on MM cells via the glycolytic pathway is still unclear. In this study, Western blotting results showed that the mechanism of PFK15's anti-myeloma activity was via suppression of the MAPK signaling pathways to trigger MM cell death. Signal Transducer and Activator of Transcription 1 (STAT1) is not only traditionally a transmitter of interferon signaling and a tumor promotor but is associated with glycolysis based on transcriptomic-proteomic expressional analysis [41]. Therefore, our study aimed to clarify the relationship between MAPKs/STAT1 and glycolysis with a particular focus on the rate-limiting enzyme PFKFB3. Metformin, which lowers glucose levels, has been confirmed by our research group to exert anti-myeloma activity via the PI3K/Akt/mTOR signaling pathway [16]. Meanwhile, we found that metformin suppressed PFKFB3 protein expression by gene chip (data not shown) and Western blotting techniques. Based on the experi- mental data above, the present study revealed a synergistic anti- myeloma effect of PFK15 with metformin in MM cell lines.
In conclusion, our study has demonstrated the mechanism of PFK15 as a PFKFB3 inhibitor, and PFK15 has anti-myeloma effects both in vitro and in vivo. However, at present available metabolic modulators are very limited and not in clinical use to date. Based on the results of PFK15 application to cancer cells, regulation of PFKFB3 with a novel drug alone or in combination with metformin may provide a precise and attractive targeted therapy for MM treatment.

Conflicts of interest

The authors declare that they have no conflict of interest.

This work was supported in part by grants from the National Natural Science Foundation of China(81201868, 81471532, 81560030 and 31371380).

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