The anti-tumor effects of the combination of microwave hyperthermia and lobaplatin against breast cancer cells in vitro and in vivo

Abstract Background: Breast cancer is the main lethal disease among females. The combination of lobaplatin and microwave hyperthermia plays a crucial role in several kinds of cancer in the clinic, but its possible mechanism in breast cancer has remained indistinct. Methods: Mouse models were used to detect breast cancer progression. Cell growth was explored with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2H-tetrazolium) and colony formation assays. Cell migration and invasion were investigated with a transwell assay. Cell apoptosis was probed with flow cytometry. The expression of apoptosis-associated proteins was examined with Western blots. Result: Combination treatment decreased breast cancer cell viability, colony formation, cell invasion and metastasis. In addition, the treatment-induced breast cancer cell apoptosis and autophagy, activated the c-Jun N-terminal kinase (JNK) signaling pathway, suppressed the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, and down-regulated IAP and Bcl-2 family protein expression. Conclusion: These results indicate that lobaplatin is an effective breast cancer anti-tumor agent. Microwave hyperthermia was a useful adjunctive treatment. Combination treatment was more efficient than any single therapy. The possible mechanism for this effect was mainly associated with activation of the JNK signaling pathway, inactivation of the AKT/mTOR signaling pathway and down-regulation of the Bcl-2 and IAP families.


Introduction
Breast cancer is the most frequently diagnosed cancer and the main cause of cancer deaths among females worldwide. Breast cancer accounts for 15% of all cancer deaths and 25% of all cancer cases among females [1]. It is therefore necessary to develop novel therapies and anti-tumor agents. Lobaplatin is a third-generation platinum (D-19466, 1,2-diamino-methyl-cyclobutane-platinum (II)-lactate) drug [2]. It is considered a potential drug for the treatment of multiple solid tumors, with encouraging anti-cancer activity and low organ toxicity [3][4][5].
Hyperthermia, raising the temperature of tumor-loaded tissue to 40-43 • C, has been applied as an adjunctive therapy with various established cancer treatments, such as radiotherapy and chemotherapy [6]. Combining hyperthermia with other cancer treatment modalities makes them more effective [7]. Co, Ltd) at 43 • C for 1 h, and kept at a temperature that fluctuated within 0.5 • C. Lobaplatin was administered once a week, while microwave hyperthermia was performed twice a week. On the 16 th day the mice were sacrificed, and their lungs were subjected to postmortem. Colonized nodule diameters were evaluated under the dissecting microscope. The total number of lung surface colonizations were recorded. Lung colonized nodules with a diameter of more than 1 mm were counted. The experiment was approved by the committee for the Humane Treatment of Animals Shanghai Jiaotong University. The animal work took place in Shanghai Jiao Tong University Laboratory Animal Center and was compliant with all of the relevant ethical regulations regarding animal research. The Experimental Animal Ethics No. A 2017054.

Flow cytometry
Cells were seeded in six-well plates and treated with lobaplatin 10 μg/ml and microwave hyperthermia at 43 • C for 1 h. After 24 h, the cells were incubated with Propidium Iodide (PI) and Annexin V for 15 min at room temperature in a dark place. The cells were then suspended and examined with flow cytometry. Apoptotic cell quantification was performed according to the Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) protocol.

Migration and invasion assay
Breast cancer 4T1 and MDA-MB-231 cells were treated with lobaplatin 10 μg/ml, microwave hyperthermia at 43 • C for 1 h and combination treatment. After being washed with PBS, cells (5 × 10 5 /100 μl DMEM) were seeded in the upper well (8-μm polycarbonate membrane, Falcon) of a 24-well plate and 500 μl of 20% FBS DMEM was added to bottom well. After incubation for 24 h, the membranes with the migrated cells were stained with 0.1% Crystal Violet. Migrated cells were photographed, counted and statistically analyzed. For the invasion assay, the upper wells with the polycarbonate membrane were coated with 60 μl of Matrigel (3 mg/ml, Becton, Dickinson and Company). Cells were seeded, treated and statistically analyzed according to the transwell migration assay protocol.

Statistical analysis
All statistical calculations were performed using analysis of variance (ANOVA). A statistical significance value of P<0.05 was regarded as statistically significant. Data are expressed as the mean + − standard deviation (SD).

The effects of lobaplatin and microwave hyperthermia on tumor cell colonization and tumor invasion
The pulmonary colonization model was set up in BALB/c mice ( Figure 1A-C). Pulmonary colonized nodules (diameter ≥ 1 mm) were counted. The greatest incidence of pulmonary colonized nodules was observed in the control group. Lobaplatin or microwave hyperthermia therapy effectively reduced the number of pulmonary colonized nodules. Furthermore, the combination of lobaplatin and microwave hyperthermia therapy significantly reduced the number of pulmonary colonized nodules (diameter ≥ 1 mm) compared with that after control treatment, lobaplatin treatment or microwave hyperthermia therapy alone. Pulmonary colonized nodules were verified with H&E staining ( Figure 1A).
An orthotopic transplant tumor model was used to study the inhibitory effects of combination therapy on tumor invasion and metastasis ( Figure 1D-I). Lobaplatin or microwave hyperthermia monotherapy suppressed tumor volume and growth. Furthermore, combination therapy significantly suppressed the tumor volume and decreased the tumor weight compared with those of the vehicle-treated control mice ( Figure 1G-I). In addition, the results showed that the combination treatment of microwave hyperthermia and lobaplatin significantly inhibited 4T1 mammary cancer cell distant metastasis compared with a single therapy ( Figure 1D-F). These results showed that the combination treatment of microwave hyperthermia and lobaplatin significantly suppressed breast cancer progression in vivo. The microwave therapy device schematic diagram was shown in Figure supplementary 5.

The combination of hyperthermia and lobaplatin induced both apoptosis and autophagy in 4T1 and MDA-MB-231 cells
Flow cytometry analysis of FITC-Annexin V/PI was used to investigate cell viability and cell death. As shown in Figure 4A, microwave hyperthermia or lobaplatin increased cell apoptosis. We hypothesized that combination therapy induced breast cancer cell apoptosis, which plays a key role in suppressing breast cancer progression. Western blots were utilized to detect the hallmarks of apoptosis [17]. The data showed that both microwave hyperthermia and lobaplatin induced PARP and Caspase 3 cleavage. Our data showed that combination treatment significantly induced more 4T1 and MDA-MB-231 cell apoptosis than that of both therapies alone ( Figure 4B-J). Furthermore, the autophagy hallmarks LC3B, P62 were detected, with increased LC3BII and decreased P62 ( Figure 4B-D). These results show that combination treatment induced both the apoptosis and autophagy of breast cancer cells.
Transwell assay was utilized to investigate the in vitro autophagy functions of breast cancer cells in migration and invasion. BFA (autophagy inhibitor) was utilized to inhibit autophagy flux. As shown in Supplementary Figure S2A  inhibited. However, our data show that BFA (10 nmol/l) has an effect on invasion, but has no effect on migration (Supplementary Figure S2C,D). The optimal BFA concentration needs further exploration.

Combination therapy with hyperthermia and lobaplatin inhibited the PI3K/AKT/mTOR signaling pathway and activated the JNK signaling pathway in 4T1 and MDA-MB-231 cells
As shown in Figure 5A-C, lobaplatin, microwave hyperthermia and combination treatment affected the PI3K/AKT/mTOR pathway. The combination of lobaplatin and microwave hyperthermia reduced the phosphorylation of mTOR, AKT, and P70S6K. Consistent with Figures 1-3, combination treatment reduced the metastasis of breast cancer in vitro and in vivo. The data indicated that lobaplatin, microwave hyperthermia and combination

The combination therapy of hyperthermia and lobaplatin suppressed the anti-apoptotic Bcl-2 and IAP proteins in 4T1 and MDA-MB-231 cells
Western blots were used to investigate Bcl-2, Bcl-xl, Mcl-1, c-IAP1, c-IAP2 and XIAP protein expression ( Figure  6). Our data showed that the combination of microwave hyperthermia and lobaplatin synergistically suppressed the anti-apoptotic Bcl-2 and IAP family of proteins in breast cancer cells. Western blots were used to investigate the pro-apoptotic effect of proteins (Supplementary Figure S4). These data indicated that microwave hyperthermia enhances the effects of lobaplatin on inducing cell death through the mitochondrial apoptosis pathway in breast cancer cells. We further investigate intracellular signaling pathways as control data, such as CyPA and HMGB1 in Supplementary Figure S6. Our data indicated that microwave hyperthermia and lobaplatin have no obvious effect on expression of HMGB1 and CyPA.

Discussion
As far as we know, the molecular mechanism by which lobaplatin kills breast cancer cells has not been clearly illustrated. In the present study, we investigated a combination therapy consisting of lobaplatin and microwave hyperthermia in vivo and in vitro. MDA-MB-231 and 4T1 were selected, for further investigation since they are highly invasive TNBC breast cancer cell lines [18,19]. The 4T1 tumor is highly tumorigenic; unlike most tumor models, it can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites [20]. We proved the combination therapy of microwave hyperthermia and lobaplatin led to synergistic anti-tumor effects in vivo. Actually, lobablatin has been confirmed as an effective anti-tumor drug on Retinoblastoma and against Ishikawa endometrial cancer cells [21,22]. We provided evidence that the combination of hyperthermia and lobaplatin strongly suppressed breast cancer cell metastases in vivo ( Figure 1B). Cancer metastasis requires the invasion of tumor cells into the stroma and then migration of tumor cells through the vasculature and lymphatics to secondary organs [23].
Then, we focused on migration and invasion. Lobaplatin has been proven to inhibit the migration, invasion and proliferation of prostate cancer cell lines [24]. In this study, we confirmed the synergistic effects of combining microwave hyperthermia and lobaplatin against breast cancer in both colony-forming and transwell experiments in vitro. Combination therapy inhibited the breast cancer cell colony-forming ability and decreased cancer invasion and migration which is better than lobaplatin monotherapy did. In 4T1 and MDA-MB-231 more cells invade than migrate (Supplementary Figure S3E,F) under combination treatment. Our data suggest that the invasion is more sensitive to treatment than migration. MTS data indicate that lobaplatin suppressed 4T1 and MDA-MB-231 proliferation depending on the timing and dosage treatment that are in-line with previous research [25]. 4T1 is more sensitive than MDA-MB-231 to lobaplatin in cell proliferation. This mainly indicated that different cells had their most optimal doses. We confirmed that the combination of microwave hyperthermia and lobaplatin better inhibited the proliferation of breast cancer cells compared with a single therapy at 24 th -, 48 th -and 72 nd -hour post treatments ( Figure 3). These data are in line with the previous experiments in vivo.
In the present study, the combination therapy consisting of microwave hyperthermia and lobaplatin was found to induce both the apoptosis and autophagy of 4T1 and MDA-MB-231 cells. We demonstrated that lobaplatin and microwave hyperthermia could induce breast cancer cell apoptosis, especially when used in synergistic treatment. Many pathways regulate both apoptosis and autophagy. It has been well accepted that the PI3K/AKT/mTOR signaling pathway plays a major role in apoptosis and autophagy [26][27][28].
Our data showed that lobaplatin, microwave hyperthermia and combination treatment inhibited the PI3K/AKT/mTOR signaling pathway. Combination treatment profoundly reduced phosphorylation of mTOR, AKT and P70S6K. MAPK family contains ERK, P38 and JNK groups, which play a key role in controlling the balance between apoptosis and autophagy [29,30]. We found that lobaplatin and microwave hyperthermia synergistically promoted the phosphorylation of JNK and P38 ( Figure 5).
It has been well documented that anti-apoptotic Bcl-2 and IAP family of proteins were indicators of apoptosis [31,32]. The Bcl-2 family contains the pro-apoptotic effector proteins Bak, Bax, Bid, Bad etc [33]. Bcl-xl and Mcl-1 at the mitochondrial outer membrane block apoptosis factors and control programmed cell death [34]. XIAP, c-IAP1 or c-IAP2 could directly inhibit caspase and impede apoptosis [35]. We further explored the anti-apoptotic Bcl-2 and IAP family of proteins in the setting of lobaplatin, microwave and combination therapy in 4T1 and MDA-MB-231 cells. In our study, lobaplatin, hyperthermia and combination treatment decreased the expression of anti-apoptotic proteins and significantly increased the levels of the pro-apoptotic proteins BAK and BAX, which play a key role in the intrinsic pathway of apoptosis [36].
Currently, the exact mechanism of combination therapy of anti-tumor efficacy needs further exploration. However, the possible mechanisms may be the following according to our finding. In the first place, combination therapy induced 4T1 and MDA-MB-231 breast cancer cell apoptosis via the activation of the JNK signaling pathway, inhibiting the AKT/mTOR signaling pathway and down-regulating the Bcl-2 and IAP family (Figure 7). It has been reported that lobaplatin inhibits PI3K/AKT/mTOR pathway, Bcl-2 and IAP family and activates members of MAPK family so does hyperthermia [24,25,29,[37][38][39][40]. Secondly, we suspected that treatments inhibiting proliferation, migration and invasion of 4T1 and MDA-MB-231 cell lines by suppressing the PI3K/AKT/mTOR pathway. This hypothesis coincides with other reports [38,41].
We further investigated other intracellular signaling pathways as control data, such as CyPA and HMGB1 in Supplementary Figure S6. Microwave hyperthermia and lobaplatin do not affect the expression of HMGB1 and CyPA. This may indicate microwave hyperthermia and lobaplatin did not have excellent synergistic effect in regulating the HMGB1-related necroptosis pathway and CyPA-related cell death pathway.
Finally, we tested the effect of combination therapy on cell pyrolysis in Supplementary Figure S7. Our data suggest that combination therapy can significantly increase the GSDMD-N cleavage (especially in 4T1 cells), indicating that combination therapy can cause cell pyroptosis significantly. Our study now, for the first time, found this phenomenon. It is still unclear whether pyroptosis was caused by microwaves or just 43 • C temperature. Compared with the results of other cell pyroptosis studies, we have too many miscellaneous bands. The death way of cancer cells caused by combination therapy needs further exploration. In summary, our data revealed that lobaplatin and microwave hyperthermia synergistically inhibited tumor growth and metastasis in murine models. Combination treatments inhibit the proliferation, invasion and migration of 4T1 and MDA-MB-231 breast cancer cells by down-regulating AKT/mTOR signaling pathway. These treatments induced apoptosis and autophagy in 4T1 and MDA-MB-231 cells via activating the JNK signaling pathway, inhibiting the AKT/mTOR signaling pathway and down-regulating the Bcl-2 and IAP family. In this article, we also focus on autophagy ( Supplementary Figures S2 and S3). However, the ideal dose of BFA is not clear. Further study is needed to explore the role of autophagy in breast cancer cell death and metastasis during combination treatment consisting of microwave hyperthermia and lobaplatin.

Data Availability
All supporting data are included within the main article and its supplementary files.

Compliance with Ethical Standards
All experiments in this manuscript comply with the current laws of China.