OSW-1 inhibited the growth of TNBC cells in vitro
Previous studies have shown that OSW-1 inhibited the growth of pancreatic cancer, hepatocellular carcinoma, colon cancer and breast cancer cells in vitro, and had lower toxicity on normal endothelial cells15–17, 19. The current study focused on the anticancer activities of OSW-1 to suppress TNBC. We selected human TNBC cell line (MDA-MB-231) and mouse TNBC cell line (4T1) for further experiments. MTT assay showed that OSW-1 strongly reduced the viabilities of both TNBC cell lines in a time-dependent manner (Fig. 1a). Colony formation was measured after OSW-1 treatment to further determine the inhibitory effects of OSW-1 on TNBC cell proliferation. The data clearly showed that OSW-1 inhibited the number and size of TNBC cell colonies at very low concentrations (Fig. 1b, c). In summary, OSW-1 significantly inhibited TNBC cell growth in vitro with very high activities in the current study.
OSW-1 induced apoptosis of TNBC cells in vitro
Inducing apoptosis is a very common mechanism of anticancer agents. OSW-1 has been shown to cause apoptosis of cancer cells. We examined the apoptosis of TNBC cell lines after OSW-1 exposure. As shown in Fig. 2a, OSW-1 treated TNBC cell lines showed the signs of cell contraction, a typical characteristic of apoptotic cells, indicating the occurrence of cell apoptosis. Hoechst 33342 staining were performed after incubating with OSW-1 for 48 h. Bright-blue fluorescent condensed nuclei, reduction of cell volume and nuclear fragmentation were observed and further showed the features of apoptosis (Fig. 2b). FCM was used to quantitatively analyze OSW-1-induced apoptosis. Treatment with OSW-1 for 48 h increased the percentage of apoptosis of 4T1 and MDA-MB-231 cells in a concentration-dependent manner (Fig. 2c, d). Expression analysis of some key proteins related to apoptosis also confirmed that OSW-1 treatment induced cleavage of caspase 3 and PARP, two hallmark events during apoptosis (Fig. 2g-i). We also found that OSW-1 reduced the expression of phosphorylated AKT (Fig. 2g-i), which is consistent with previous studies29. In summary, these findings show that the inhibitory activity of OSW-1 on TNBC cells occurs through apoptosis.
Osw-1 Likely Induced Apoptosis Via The Mitochondria-mediated Intrinsic Pathway
We determined the expression levels of some apoptosis-related proteins to explore the mechanism of apoptosis. Bcl-2 family proteins play pivotal roles in apoptosis by maintaining mitochondrial homeostasis. Previous studies reported that Bcl-2 family proteins maintain balance between anti-apoptotic and pro-apoptotic proteins, which are crucial for regulating mitochondrial integrity30. Expression of Bcl-2 family proteins in TNBC cells was determined after treatment with OSW-1 for 48h and the data showed that OSW-1 increased the expression of Bax and decreased that of Bcl-2, leading to significant increase in Bax/Bcl-2 expression ratio (Fig. 2g-i). These findings suggest that OSW-1 might induce apoptosis through the mitochondria-mediated intrinsic pathways. To verify this hypothesis, we determined the changes of ΔΨm by FCM after OSW-1 treatment using Rh123. Our findings showed that OSW-1 caused loss of ΔΨm in 4T1 and MDA-MB-231 cells (Fig. 2e, f). Together, our data indicate that mitochondria-mediated intrinsic apoptotic pathway plays important roles in OSW-1-induced cell death.
OSW-1 induced cyto-protective autophagy and impared autophagy flux in TNBC cells
Autophagy is a double-edged sword in cancer progression and cancer treatment. Previous studies showed that OSW-1 treatment activated autophagy in colorectal cancer and pancreatic cancer cells31. To elucidate the role of autophagy in OSW-1 induced TNBC cell death, we conducted several assays. Conversion of LC3-I into lipid LC3-II and formation of LC3 puncta are regarded as two biomarkers of autophagy. The current study established that OSW-1 treatment induced increase in LC3-II expression (Fig. 3a) and accumulation of LC3 puncta (Fig. 3b, c), indicating activation of autophagy. Expression level of autophagy-specific substrate p62 is an indicator of autophagy flux, which is involved in autophagy regulation and maintains intracellular homeostasis and signal transduction. We observed that p62 levels increased in TNBC cells after OSW-1 treatment (Fig. 3a), suggesting that OSW-1 might impair autophagy flux. To investigate whether the impaired autophagic flux is due to failure in autophagosome-lysosome fusion, a tandem RFP-GFP-labeled LC3B construct was used. The data showed that most LC3B puncta showed RFP+GFP+ signals (autophagosomes) rather than RFP+GFP− signals (autolysosomes) (Fig. 3d-f), indicating that OSW-1 treatment caused accumulation of autophagosomes and decreased autolysosome formation. The above data showed that OSW-1 blocked autophagy flux in TNBC cells. To elucidate how autophagy mediated the anti-TNBC effects of OSW-1, MDA-MB-231 and 4T1 cells were treated with OSW-1 in combination with chloroquine (CQ), an autophagosome-lysosome fusion inhibitor, or Wortmannin (WORT), a PI3K inhibitor which prevent autophagy initiation and autophagosome formation. The data clearly showed that OSW-1 induced cell growth suppression was significantly enhanced after combination with CQ and Wortmannin in a time-dependent manner. Overall, these data showed that OSW-1 induced protective autophagy and inhibition of autophagy could aggravate OSW-1’s anti-cancer activities in TNBC cells.
OSW-1 sensitizes TNBC cells to DOX treatment in vitro
DOX is the first-line drug for TNBC chemotherapy due to its excellent anticancer efficacies. However, the dose-dependent cardiotoxicities restricted its clinical use and should not be neglected. Therefore, it’s of great significance to find effective combination strategies to increase the anticancer efficacies of DOX. We explored whether OSW-1 enhances the antitumor effects of DOX. MTT assay was undertaken to determine the effects of combination of OSW-1 and DOX in TNBC cells. Three different concentrations of each drug were selected for the permutation and combination. Our results showed that OSW-1 and DOX synergistically inhibited the viabilities of 4T1 and MDA-MB-231 cells (Fig. 4a). To quantitatively evaluate the synergy between DOX and OSW-1, combination index was calculated using Compusyn software (Fig. 4b). The data demonstrated that the CI points were below 1 in all selected concentration combinations. Moreover, very strong synergistic effects (CI < 0.1) were observed at certain concentrations. Colony formation assay was conducted to further investigate the synergistic effects of OSW-1 and DOX and the drug combinations exhibited better inhibition than either monotherapy (Fig. 4c, d). These data suggested a synergistic effect of DOX and OSW-1 to inhibit TNBC cell viabilities.
Both OSW-1 and DOX exert anti-TNBC effects by inducing apoptosis32. We speculated that the synergistic effect of DOX and OSW-1 also resulted from enhanced apoptosis. Accumulation of apoptotic cells was determined by Annexin V/7-AAD staining after treatment with OSW-1, DOX and their combination for 48 h. The proportion of apoptotic cells was 15.67% after OSW-1 monotherapy and 10.99% after DOX monotherapy in 4T1 cells. Notably, the apoptotic rate in combination treatment group was increased to 48.6%, which was nearly 3–4 times of that in monotherapy group (Fig. 4e, f). A similar trend was observed in MDA-MB-231 cells (Fig. 4e, f).
Destruction of ΔΨm is one of earliest intracellular events during intrinsic apoptosis. We found that combination treatment caused more than four times of ΔΨm loss when compared to either DOX or OSW-1 alone, suggesting that the dual treatment might enhanced intrinsic apoptosis (Fig. 4g, h). In addition, the enhanced suppressive effects from combination treatment was accompanied by up-regulation of cleaved caspase 3, down-regulation of phosphorylated AKT, and up-regulation of the Bax/Bcl-2 expression ratio. Together, our data indicated that OSW-1 effectively enhances DOX-induced TNBC suppression.
OSW-1 sensitizes TNBC tumor growth and spontaneous metastasis to DOX treatment in vivo
Having demonstrated that OSW-1 enhanced the inhibitory of DOX in vitro, we next sought to determine the in vivo effects of this combination. We generated 4T1 TNBC models in vivo by subcutaneous inoculation of the cells into BALB/c mice. Notably, both OSW-1 and DOX modestly inhibited tumor growth in vivo (Fig. 5a), while combined DOX/OSW-1 treatment showed the best inhibitory effects on tumor growth without causing obvious weight loss of the animals (Fig. 5a, b). To determine the effects of the drugs on cancer cell proliferation and apoptosis in vivo, tumor tissue was stained with Ki-67 and cleaved caspase 3. We found that the abundance of Ki-67 was decreased most while the staining score of cleaved caspase 3 was the strongest in the combination treatment (Fig. 5c, d).
Metastasis is the main reason of cancer related death. Spontaneous lung metastasis and systemic metastasis in mice were evaluated in the current study because lung is a major metastatic site of TNBC. The primary 4T1 tumors were surgically removed from the mice, after which the metastases were visualized using IVIS imaging. While lung metastasis was inhibited by OSW-1 and DOX monotherapy, the combination of the two agents exhibited the strongest inhibition (Fig. 5e). Additionally, the combination treatment exerted better inhibition of systemic metastasis than monotherapy as evidenced by the quantification of luminescence intensity. Together, our data demonstrate that the addition of OSW-1 augmented the anticancer effects of DOX, especially in suppressing metastasis (Fig. 5f).
Combination of OSW-1 and DOX promoted the infiltration of CD8 + T cell in the lung
The immune microenvironment plays critical roles in cancer metastasis and significantly influences treatment response and overall outcome of cancer patients. It has been shown that DOX could regulate the infiltration of some key immune cells in the TME33. Since immune cells play vital roles in cancer metastasis, we speculated that regulating immune cells in the TME are involved in the inhibition of metastasis.
CD8+ T cells play important roles in adaptive cancer immunity to eliminate cancer cells. The efficacy of most immunotherapy depends on the activities of CD8+ T cells. Cytotoxic CD8+ T cells entered tumor microenvironment, and exhibited cytotoxicities to tumor cells34. CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function35. While neither DOX nor OSW-1 showed obvious effects on the proportion of CD8+ T cells in the lung, we found that its frequencies were augmented after combination treatment (Fig. 6a). We also detected the expression of the activation marker (CD69) and exhaustion marker (PD-1) on CD8+ T cells. The data showed that its activation was not influenced after treatment. While DOX decreased the expression of PD-1 to some extent (p > 0.05), the addition of OSW-1 did not shown synergistic effects (Fig. 6g, h). CD4+ T helper cells are another group of key regulators of anticancer immunity in TME, which mediate adaptive immune response to cancer mainly by activating cytotoxic CD8+ T cells34. DOX treatment increased the proportions of CD4+ T cells in the lung. However, we didn’t see a synergism between DOX and OSW-1 to influence the frequencies of CD4+ T cells (Fig. 6b).
Neutrophils mainly promote cancer progression through several mechanisms, including promoting angiogenesis, immunosuppression and metastasis36. Targeting neutrophils represent a promising anticancer strategy. We observed that combination of OSW-1 and DOX significantly reduced the frequencies of neutrophils among CD45+ leukocytes compared with vehicle and OSW-1 monotherapy (Fig. 6c). However, no significant differences were observed between OSW-1 and DOX groups, indicating that the addition of OSW-1 did not affect the effects of DOX.
Dendritic cells (DCs) are the most powerful antigen-presenting cells and play central roles in regulating the balance between CD8+ T cell immunity and tolerance to tumor antigens37. Although DOX treatment increased the frequencies of DCs in the lung, there were no significant differences between the combination treatment and DOX treatment group.
Macrophages play complicated roles in the progression of lung metastasis. Among them, M2 (CD206+) subtypes play promoting role in more cases38, 39. In this study, we showed that DOX monotherapy caused a non-significant increase in the percentage of total F4/80+ macrophages in leukocytes. Intestinally, DOX treatment significantly decreased the proportions of M2 subtypes. Notably, OSW-1 treatment did not affect the effects of DOX to influence the number of macrophages. In summary, our data suggest that CD8+ T might play important roles in the suppression of lung metastasis from the combination treatment. Notably, the addition of OSW-1 didn’t exert inhibitory effects to DOX’s anticancer immunity.