Lanthanum Chloride Sensitizes Cisplatin Resistance of Ovarian Cancer Cells via PI3K/Akt Pathway

Our previous study manifested that lanthanum chloride (LaCl3) can enhance the anticancer ability of cisplatin (DDP) in ovarian cancer cells. Here, ovarian cancer cells SKOV3 and SKOV3/DDP were subjected to DDP and LaCl3. Cell viability, apoptosis, DNA repair, and PI3K/Akt pathway were detected. LaCl3 induced more cell death and apoptosis caused by DDP in two cell lines, accompanied by upregulation of Bax and Cleaved caspase 3 proteins, and downregulation of Bcl-2 protein. LaCl3 also could decrease RAD51 protein by inactivation of the PI3K/Akt pathway. These data indicated that LaCl3 could be a potential drug to modulate DDP resistance by inactivating of PI3K/Akt pathway and attenuating DNA repair in ovarian cancer.


INTRODUCTION
Ovarian cancer is the most lethal gynecologic cancer. The first-line treatment for ovarian cancer is cisplatin (DDP)-based chemotherapy after cytoreductive surgery. However, the DDP resistance of ovarian cancer during the treatment is an important reason for the treatment failure. The 5-years survival rate is <40% (1,2).
Cisplatin (DDP) often attacks DNA to cause DNA damage and lead to cell apoptosis; therefore, an enhanced DNA repair plays a key role in DDP resistance (3,4). Survival pathways were necessary for cell survival and involved in chemoresistance. The PI3K/Akt survival pathway was a way to play an important role in cell survival and DDP resistance in ovarian cancer. Akt can activate the cyclin D1, NF-kB, mTOR, RAD51 or inhibit the caspase 9, p21, p27 to inhibit cell apoptosis (5)(6)(7). Lots of research target the DNA repair and PI3K/Akt pathway to explore new drugs to reverse the DDP resistance and improve the prognosis of ovarian cancer (8,9). However, there are still no effective therapies due to the side effect. Therefore, it is important to explore novel drugs to conquer the resistance of DDP.
Lanthanum chloride (LaCl 3 ) is a complex of rare earth elements. Recent studies have reported that LaCl 3 can inhibit the proliferation and migration of cancer cells and can be an effective drug to kill cancer cells (10)(11)(12). Our previous study indicated that LaCl 3 could augment the anticancer ability of DDP (13,14). However, the mechanisms of LaCl 3 acts on ovarian cancer remain unclear.
Research showed that LaCl 3 can downregulate the PI3K/Akt signaling pathway to cause cytotoxicity; and LaCl 3 can inactivate the Akt signaling pathway to induce autophagy (15, 16). Considering the important role of the PI3K/ Akt pathway in DDP resistance. We suggest that LaCl 3 may reverse DDP resistance via PI3K/Akt pathway. Therefore, in this study, the relationship of LaCl 3 and PI3K/Akt was explored using DDP sensitive and DDP resistance ovarian cancer cells.
Preliminary data showed that LaCl 3 could inactivate the PI3K/Akt pathway to inhibit DNA repair, eventually enhancing the antitumor ability of DDP in ovarian cancer.

Cell Apoptosis
Apoptosis cells were detected using an Annexin V assay (Keygen Biotech., Nanjing, China) after Cells were treated with DDP (IC 50 ) and/or LaCl 3 (1.5 µmol/L) for 48 h.

Activated Assay
Cells were exposed to SC79 (an activator of Akt: 4 µg/mL;) (MedChemExpress) co-culture with DDP (IC 50 ) and/or LaCl 3 (1.5 µmol/L) for 48 h. Then CCK8 was used to detect the survival cells, and western blot was used to detect the RAD51 protein.

Statistics
Data were processed with the software SPSS 26 (IBM, Armonk, United States). ANOVA and t-test were performed. The difference was significant if p < 0.05.

LaCl 3 Enhanced the Efficacy of DDP in Ovarian Cancer Cells
The IC 50 values of DDP were 5 and 15 µmol/L for SKOV3 and SKOV3/DDP cells, respectively, confirming the resistance phenotype of SKOV3/DDP (Figure 1A). The percentages of survival cells were more than 90% in SKOV3 and SKOV3/DDP cells after exposure to LaCl 3 (0.5, 1, and 1.5 µmol/L). However, the percentage of survival cells was <90% following exposure to higher concentrations of LaCl 3 (≥ 2 µmol/L), and the percentage FIGURE 2 | LaCl 3 promoted cell apoptosis caused by DDP in ovarian cancer cells (n = 3). Apoptosis was detected by the annexin V assay; the apoptotic percentage in SKOV3 cells was increased after DDP combined with LaCl 3 (A,B). The apoptotic percentage in SKOV3/DDP cells was increased after DDP combined with LaCl 3 (A,C). *p < 0.05. of dead cells in SKOV3/DDP cells was higher than in SKOV3 (p =0.032, p = 0.001) ( Figure 1B). Therefore, 1.5 µmol/L of LaCl 3 was used in the subsequent experiments.
The percentage of the dead cells was increased in SKOV3 cells following exposure to DDP combined with LaCl 3 compared with DDP alone (p = 0.001-0.025) ( Figure 1C). In SKOV3/DDP cells, we observed the same results (p = 0.013-0.026) (Figure 1D). These results indicated that LaCl 3 enhanced the cytotoxicity of DDP.

LaCl 3 Enhanced Apoptosis Due to DDP
DDP caused SKOV3 and SKOV3/DDP cell apoptosis, and the combination of LaCl 3 and DDP led to a higher percentage of apoptotic SKOV3 and SKOV3/DDP cells (p = 0.005, p = 0.001) (Figure 2).

LaCl 3 Inhibited DNA Repair by PI3K/Akt Pathway
DNA repair was assayed by detecting RAD51 since RAD51 is a key molecule for homologous recombination (HR) (19). Recent studies indicated that the PI3K/Akt can induce the expression of RAD51 (7,20). Western blot was used to detect the expression of RAD51 and PI3K/Akt. DDP increased the RAD51 level, which means DDP not only induced DNA damage but also initiated DNA repair. However, LaCl 3 decreased the level of RAD51 in both cell lines (p = 0.002, p = 0.004) (Figures 4A,B). DDP induced the phosphorylation of PI3K/Akt, the levels of p-PI3K and p-Akt were decreased in two cell lines following LaCl 3 exposure (p = 0.04, p = 0.042, p = 0.011, p = 0.001) (Figures 4C,D).
To further demonstrate the action of PI3K/Akt on RAD51, the SC79 (an activator of Akt) was added in SKOV3 and SKOV3/DDP cells. The level of RAD51 was upregulated in both cells after exposure to SC79 (p = 0.045, p = 0.011) (Figures 5A,B). The percentages of survival cells were increased after exposure to SC79 in SKOV3 and SKOV3/DDP cells (p = 0.034, p = 0.035) (Figures 5C,D). The data indicated that LaCl 3 could inhibit DNA repair via PI3K/Akt pathway and promote the action of DDP.

DISCUSSION
DDP resistance is a key obstacle for the treatment of ovarian cancer. Hence, it is of particular importance to explore new drugs to reverse DDP resistance. Here, we demonstrated that LaCl 3 (≥2 µmol/L) caused cell death in SKOV3 and SKOV3/DDP cells. Interestingly, the percentage of dead cells in SKOV3/DDP cells was higher than in SKOV3 (p = 0.032, p = 0.001), and this needs further study to validate that the LaCl 3 was more lethal on DDP resistant cells in the future. However, the lower concentration of LaCl 3 (1.5 µmol/L) that was unharmful to ovarian cancer cells could increase cell death due to DDP. This was consistent with our previous study that LaCl 3 can enhance the cytotoxicity of DDP in ovarian cancer cells (13). Cytotoxicity of DDP was regulated by apoptosis (21,22). Hence, enhancing cell apoptosis was the main target for drugs to reverse DDP resistance (23). The combination of DDP and LaCl 3 led to the highest apoptotic percentage in SKOV3 and SKOV3/DDP cells. Then, the Bax and Cleaved caspase 3 were most expressed in both cells following DDP and LaCl 3 exposure, and Bcl-2 was the least. These were consistent with the previous study that LaCl 3 can regulate the protein expressions of Akt, Bcl-2, Bcl-xl, Bax, Bad, caspase-3, and caspase-9 to promote cell apoptosis (24); and indicated that LaCl 3 promote cell apoptosis caused by DDP to conquer DDP resistance.
The main target of DDP is DNA, DDP attacks DNA to cause a break, and unrepair damage leads to apoptosis. RAD51 is the key protein of HR for repairing DNA damage (25,26). In this study, the level of RAD51 was upregulated following DDP exposure, which meant DDP inducing DSB, then starting DNA repair. This possibly explains the reason that the cytotoxicity of DDP was decreased during treatment and eventually led to DDP resistance. However, the RAD51 level was decreased after LaCl 3 exposure. These were consistent with the results of the percentages of dead and apoptotic cells following DDP and LaCl 3 exposure and consistent with previous studies that conquer DDP resistance by inhibiting DNA repair (27). Our results primarily indicated that LaCl 3 can enhance apoptosis by inhibiting DNA repair.
The PI3K/Akt pathway is an important survival pathway that is involved in DDP resistance in ovarian cancer (28). Here, the phosphorylation of PI3K and Akt was activated by DDP, but the inductive effect of DDP was attenuated after LaCl 3 exposure. These results were consistent with previous studies that LaCl 3 can inhibit the PI3K/Akt pathway to cause cytotoxicity (15, 16). Activation of Akt can increase the expression of RAD51, while inactivation of Akt downregulates the level of RAD51 to enhance cell apoptosis caused by DNA-damaging drugs (29). Hence, the SC79 (an activator of Akt) was added to neutralize the inhibiting effect of LaCl 3 , the level of RAD51 was increased that caused by DDP combined with LaCl 3 exposure, while the percentages of dead cells were deceased. The data demonstrated that LaCl 3 inactivated PI3K/Akt pathway to downregulate the expression of RAD51.
In conclusion, the LaCl 3 could attenuate the DDP resistance of ovarian cancer cells via inhibiting PI3K/Akt pathway, downregulated RAD51 to inhibit DNA repair, and eventually promoted cell apoptosis due to DDP. Thus, LaCl 3 can be a potential drug for the treatment of ovarian cancer and DDP resistance.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.