Ph-ph+ inhibited migration and invasion of breast cancer cells
After ph-ph+ was incubated with human breast cancer cell MDA-MB-231, the cell viability dropped in concentration- and time-dependent patterns (Fig. 1a). Then the reduction was examined in another breast cancer cell SKBR-3 (Fig. 1b), suggesting that the effect of ph-ph+ against breast cancer cells might not be related to the cell heterogeneity of breast cancers. However, the same concentration of ph-ph+ did not influence the viability of human liver and kidney cells, showing specific anti-cancer activity (Fig. 1c).
In cell scratch assay, the two types of cancer cells, SKBR-3 and MDA-MB-23 cells, exhibited high migration rate within 24 h incubation (37.5% for SKBR-3 cells, 45.4% for MDA-MB-23 cells) (Fig. 1d, e). However, the migration rates in both cells were significantly reduced after 0.5 µg/mL ph-ph+ addition into the media (Fig. 1d, e), representing that the cell migration could be blocked by ph-ph+. Moreover, the transwell migration assay identified the anti-migration effect of ph-ph+ on the cancer cells (Fig. 1f). Meanwhile, transwell invasion test was conducted to test the activity of ph-ph+ against the cancer cell invasion. The results were shown in Fig. 1g. The cell invasion ability remarkably decreased from 0.5 µg/mL ph-ph+ as compared with the high invasion of the control cells. The results proved that ph-ph+ could suppress the migration and invasion of malignant cancer cells.
Ph-ph+ prevented cell proliferation of breast cancer and induced apoptosis
Cell clone formation was examined to estimate the action of ph-ph+ on rapid proliferation of malignant cells. Both ph-ph+-treated cancer cells exhibited low colony-forming efficiency (Fig. 2a, 2b) relative to the control cells, indicating that the compound could prevent cancer cell proliferation.
Under microscope, breast cancer cells treated with 4 µg/mL ph-ph+ became shrink and round, showing the apoptotic features (Fig. 2c). However, the normal cells with ph-ph+ did not show obvious changes. To further identify whether ph-ph+ could cause cancer cell apoptosis, 4,6-diamino-2-phenylindole/propidium iodide (DAPI/PI) fluorescence dye and flow cytometry were respectively applied to detect apoptotic cells. The results were shown in Fig. 2d and 2e. After the DAPI/PI staining, the cells incubated with 2 µg/mL ph-ph+ displayed red fluorescence, and more fluorescence-labeled cells appeared after 4 µg/mL ph-ph+ addition (Fig. 2d). Then the cells were detected in the flow cytometry (Fig. 2e), and the result identified that ph-ph+ could cause cancer cell apoptosis in concentration-dependent behavior.
Downregulation of PLAGL2 would participate in anticancer effect of ph-ph+
Pleomorphic adenoma gene like 2 (PLAGL2) is a nuclear transcription factor that highly expressed in various cancers such as breast cancer, colorectal cancer and gastric cancer (Additional file 1: Figure S1) [18–20]. The oncogenic property of PLAGL2 stimulates cancer cell proliferation, invasion, migration, and anti-apoptosis [21, 22]. Also, PLAGL2 would be responsible for systemic metastasis and poor prognosis in the patients [23], evaluated by bioinformatics analysis (Additional file 1: Figure S2). Here PLAGL2 target was explored to elucidate the anticancer mechanism of ph-ph+.
After the breast cancer cells incubated with ph-ph+, PLAGL2 protein expression significantly decreased, and correspondingly, of its downstream signal proteins that promote cell proliferation, invasion and migration, β-catenin and Wnt, reduced (Fig. 3a, 3b). Moreover, levels of pro-apoptotic proteins, caspase 3 and Bax, elevated, whereas the content of anti-apoptotic protein Bcl-2 decreased (Fig. 3a, 3b).
To test the role of PLAGL2 in tumor inhibition of ph-ph+, a plasmid containing fusion gene of human PLAGL2 gene and GFP gene (pPLAGL2/GFP) was constructed (Fig. 3c, Additional file 1: Figure S3). As compared with the breast cancer cells, liver cells and HEK293 cells that were not sensitive to ph-ph+ displayed weak protein expression of PLAGL2, despite the cells had the same PLAGL2 gene in their genomes as cancer cells (Fig. 3d, 3e). Thus the pPLAGL2/GFP was respectively transfected into the liver cells and HEK293 cells to obtain the transgenic cells with high expression PLAGL2. After pPLAGL2/GFP transfection, the both cells exhibited strong green fluorescence and highly expressed PLAGL2 protein (Fig. 3f, 3g). The migration and invasion of transgenic cells were blocked following 0.5 µg/mL ph-ph+ addition (Fig. 3h, 3i). When 4 µg/mL ph-ph+ incubation, the PLAGL2 transgenic cells appeared obvious apoptosis features such as cell shrinkage and rounding (Fig. 3j, 3k), suggesting that PLAGL2 would be a target in ph-ph+ treatment.
Ph-ph+ activated CLpP in mitochondria of breast cancer cells
The previous studies suggested that ph-ph+ could damage mitochondrial respiratory chain function in cancer cells and cause cellular energy insufficiency. The mechanism remains unclear. To clarify the reason for ph-ph+ on mitochondria, the mitochondrial function of breast cancer cells was examined after ph-ph+ administration, and the mitochondria were observed with transmission electron microscope (TEM). Biochemical measurement showed that the intracellular green fluorescence of JC-1 remarkably increased after ph-ph+ addition (Fig. 4a), suggesting that MMP reduced and mitochondrial structure was damaged. Also, the activities of mitochondrial electron transport chain (ETC) complexes and ATP concentration of SKBR-3 cells significantly decreased (Fig. 4b, 4c). Under TEM, the number of mitochondrial cristae reduced, and even the mitochondria appeared large vacuoles (Fig. 4d), implying a proteolytic enzyme in the mitochondrial matrix might be activated to digest the mitochondrial internal proteins.
In mitochondrial matrix, caseinolytic peptidase P (CLpP) is an evolutionarily conserved proteolytic enzyme that widely exists in mitochondria of eukaryotic cells [24–26]. Similar to PLAGL2, CLpP is highly expressed in tumor cells to degrade the misfolded and damaged proteins under physiological conditions [27]. It’s reported that activation of CLpP by chemicals can indifferently degrade mitochondrial proteins. As CLpP is activated in tumor cells, the mitochondria showed vacuoles and then energy production insufficiency [28], which was consistent with the mitochondria of ph-ph+-treated cells. Thus we speculated that ph-ph+ might damage mitochondria by CLpP activation. In the study, the CLpP activity was measured as reported, and the results were shown in Fig. 4e. The CLpP activity in breast cancer cells significantly increased following ph-ph+ addition, and exhibited time- and concentration-dependent patterns (Fig. 4e). However, the activity in liver cells and HEK293 cells only showed a slow-increasing trend, which may be partly related to the low level of CLpP protein in the cells (Fig. 4f, Additional file 1: Figure S4).
Furthermore, the molecular docking suggested that ph-ph+ could bind to the active center of CLpP, where ph-ph+ interacted with isoleucine (Ile), leucine (Leu), and valine (Val) by hydrogen bond and hydrophobic bond (Fig. 4g). The interaction of ph-ph+ and CLpP stimulated the enzyme activity, and then the activated CLpP catalyzed to indiscriminately degrade mitochondrial proteins, leading to mitochondrial dysfunction.
Ph-ph+ inhibited C. albicans reproduction by activating CLpP
To evaluate the anti-fungal action of ph-ph+ on C. albicans, MIC and MFC were examined through microtiter plate method in accord with CLSI. The results indicated that MIC and MFC were respectively presented as 2 µg/mL and 8 µg/mL.
C. albicans has two phenotypes, yeast and mycelium [29]. Yeast type of C. albicans can transform to mycelium type under physiological conditions for rapid reproduction and invasion through germination [30]. Biofilm formed by a large number of mycelia can protect the pathogenic fungus from external damage, leading to drug resistance in C. albicans [31]. Here inhibitory effect of ph-ph+ on both yeast and mycelia of C. albicans was tested. As shown in Fig. 5a and 5b, the spore germination rate of C. albicans was prevented from 2 µg/mL ph-ph+. The yeast and mycelia showed similar morphological changes of cellular content degradation observed by TEM after ph-ph+ treatment, suggesting that ph-ph+ could kill the fungus by activation of intracellular proteases. Then ph-ph+ was applied to the white velum of C. albicans mycelia, the results confirmed that ph-ph+ had the ability to eliminate the fungus and destroy the biofilm (Fig. 5c). And also, inhibitory rate of ph-ph+ against biofilm formation enhanced in concentration-dependent pattern (Fig. 5d).
To evaluate whether mitochondria of C. albicans were damaged by ph-ph+, the yeast and mycelia were respectively stained by JC-1, and meanwhile, the mitochondrial respiratory function was tested. The images showed that amounts of green fluorescence appeared in yeast and mycelia treated by ph-ph+ after JC-1 staining (Fig. 5e), and correspondingly, activities of mitochondrial ETC complexes, as well as ATP concentration, remarkably decreased (Fig. 5f, 5g).
Because the PLAGL2 gene was not present in C. albicans (Additional file 1: Figure S5), CLpP activity was measured to elucidate ph-ph+ anti-fungal mechanism. As expected, CLpP activity dramatically elevated as ph-ph+ incubated with the fungus (Fig. 5h). It’s reported that CLpP activation can promote cell stress and then induce apoptosis [27], then the apoptosis in C. albicans was detected in the study. After AnnexinV-FITC/PI staining, the red fluorescence gradually elevated as ph-ph+ concentration increased (Fig. 5i), indicating that the cells underwent apoptosis. Also, the expression of pro-apoptotic proteins, caspase 3 and caspase 9, increased, and of anti-apoptotic proteins, Bcl-xL and Bcl-2, reduced (Fig. 5j, 5k). Collectedly, the results suggested that ph-ph+ would kill C. albicans through CLpP activation-induced cell apoptosis.
Ph-ph+ cured systemic and local candidiasis
C. Albicans can cause systemic and local infections in animal bodies. During systemic candidiasis, the fungus can rapidly proliferate in various tissues, manifested as mycosis, endocarditis, meningitis, and focal lesions of the liver, spleen, kidney and other tissues [32]. Here anti-fungal action of ph-ph+ was tested in animals with systemic candidiasis (Fig. 6a). After infection of C. Albicans, the control mice without drug treatment exhibited weakness, reduced activity, weight loss, and the symptoms became obvious over days (Fig. 6b, 6c). All mice in model group were euthanized in 15 days. However, ph-ph+-treated mice only showed relatively slight symptoms, and all mice (100%) survived in the experimental period. Additionally, mice in FLC-treated group presented a lower survival rate (80%) than those injected with ph-ph+ (Fig. 6c).
After euthanasia of mice, the tissues were separated and sectioned, and then the slices were respectively dyed with periodic acid-schiff (PAS) and hematoxylin- eosin (HE). White colonies and plaques could be observed on the surface of liver and kidney tissues in the model mice, whereas the number of colonies obviously reduced after treatment of ph-ph+ and FLC (Fig. 6d). Then the fungal numbers in tissue homogenization were further determined by in vitro fungal culture. A large number of fungi were situated in the kidney, liver, heart, lung, spleen, heart and brain of the model mice, while the number significantly decreased after ph-ph+ or FLC administration (Fig. 6e). Also, the fungal load in the kidney, liver, heart, lung and spleen of ph-ph+ treatment group was lower than those of FLC group, and no fungi was observed in heart and brain after ph-ph+ injection. Moreover, the morphology of fungi in tissues is observed by PAS staining, and the images showed that a large amount of mycelia appeared in model mouse tissues, but decreased in the treatment group (Fig. 6f). Only a relatively small amount of mycelia distributed in liver of ph-ph+ group as compared with the FLC group. Meanwhile, HE staining showed that ph-ph+ treatment could significantly alleviate inflammation induced by the fungus in the kidney and liver of mice (Additional file 1: Figure S6).
Moreover, the therapeutic effect of ph-ph+ against candidiasis was confirmed in the local C. Albicans infection of animals. Local infections often occur in many areas including the oral cavity, skin, and vagina. Oral candidiasis is a common disease in immunocompromised people [33]. Here the oral candidiasis animal model was established by applying C. albicans onto the mouse tongue (Additional file 1: Figure S7a). On the third day after application, mice in model group showed white colonies and plaques on tongue surface, and the plaques obviously increased over time (Additional file 1: Figure S7b, 7c), accompanied by decreased body weight of the mice (Additional file 1: Figure S7d). However, the symptoms were much improved after ph-ph+ administration for 4 days, and nearly disappeared following 6 days’ treatment of ph-ph+. When the tissue was homogenized and the fungus was cultured in the solid media, it was found that the number of C. albicans dramatically decreased in ph-ph+-treated mice compared to the model group (Additional file 1: Figure S7e, 7f). HE staining presented that tongue surface in the model mice was rough, mucosa thickened, and inflammatory cells infiltrated. However, ph-ph+ eliminated the fungus and exhibited a stronger antifungal effect than the positive drug FLC at the same dosage (Additional file 1: Figure S7g).
Treatment of breast cancer combination with C. albicans infection by ph-ph+
After the cancer cells were transplanted to mouse right breast, tumor mass began to rapidly grow at the 7th day. As the tumor size arrived to 100 mm3, C. Albicans was intravenously injected into the mice (Fig. 7a). Then mice were assigned to three groups. Tumor mass in model mice that received only saline continued to grow and mouse body weight decreased (Fig. 7b, 7c). However, tumor growth became slow in the treatment groups of ph-ph+ and positive control (cyclophosphamide and FLC combination therapy, CTX + FLC), and body weight in ph-ph+ group had not obvious reduction compared to before ph-ph+ administration. After the tumor tissues were respectively separated, it was found that the mouse tumor weight and size in the ph-ph+-treated group remarkably decreased relative to the model group and combination therapy group (Fig. 7d, 7e). HE staining exhibited that tumor tissue in model group was a dense region with tumor cells, whereas tumor cells in the ph-ph+ treatment group were relatively sparse and the tumor area scattered with cell fragments that were not stained with hematoxylin (Fig. 7f). The cell apoptosis assay by TUNEL staining suggested that numerous apoptotic cells appeared in tumor tissue following ph-ph+ administration (Fig. 7g), indicating that ph-ph+ inhibited tumor growth through inducing cell apoptosis.
Moreover, the fungi within mouse kidney, liver and tumor tissues were respectively detected by fungal culture of tissue homogenates. It’s shown that ph-ph+ could significantly decreased the fungal load in tissues, and its anti-fungal effect was superior to the CTX and FLC combination therapy group (Fig. 7h).
Ph-ph+ inhibited breast cancer metastasis in animals
In the model mice of breast cancer combined with candidiasis, the tumor masses were found in lymph nodes, intestine, liver, lung with 100% metastasis rate. Metastatic breast cancer formed solid tumor nodules of different sizes scattered in the intestine of the model animals (Fig. 8a). Despite CTX and FLC combination therapy could reduce the tumor size, no significant difference presented in tumor number between model group and combination therapy group. However, only very small tumor cell protrusions were observed in the intestine following ph-ph+ treatment, and meanwhile, the tumor number dropped dramatically (Fig. 8a).
Liver is a common metastatic place in the breast cancer patients, and liver metastasis represents a poor survival rate compared to other metastatic tissues [34–36]. In the animal model, obvious tumor mass could be observed on the liver surface (Fig. 8b). After tissue sectioning and HE staining, large tumor nodules filled with tumor cells were shown in the liver slices of model mice (Fig. 8c). The tumor nodules became small and tumor cell numbers decreased after the combination therapy group. However, the tumor nodules almost completely disappeared in ph-ph+-treated group (Fig. 8c). Similarly, the anti-metastasis effect of ph-ph+ also presented in lung and lymphatic metastasis of breast cancer by tissue sectioning and staining (Fig. 8d, 8e).