Synthesis of by241

As shown in Fig. 1, the synthesis of by241 involved the vinylogous aldol reaction of the steroidal dicyanoalkyene 1 with isatin, followed by intramolecular cyclization and isomerization sequence in the presence of Et 3 N, affording by241 in 88% yield under mild conditions. One C-C single bond, one C-O bond, as well as one quaternary carbon center were formed in this one-pot reaction. It should be noted that we obtained intermediate B from compound 1 when this kind of reaction was performed in EtOH, not the EtOH/H 2 O mixture using the sterically hindered DBU as the base. The structural characteristics of by241 lie in the spiro-fused oxindole and 2H-pyran scaffolds connected through a quaternary carbon center. Compound 1 was efficiently prepared from DHEA within two steps following our previously reported methods18.

Cytotoxicity of by241 against human cancer and normal cell lines

With by241 in hand, we next tested its cytotoxicity against several human cancer cell lines of different origins using the MTT assay. The well-known anticancer drug 5-fluorouracil (5-FU) was selected as the reference drug to compare the in vitro and in vivo anticancer potency. Human normal liver cell line (L-02) and human normal esophageal cell line (Het-1A) were chose to investigate the toxicity and selectivity of by241. The IC 50 values of by241 and 5-FU against tested cancer cell lines and human normal cell lines are summarized in Table 1. Generally, by241 had broad-spectrum anticancer activity, showing favorable inhibition against the tested cancer cell lines (IC 50 < 6.5 μM). Also, by241 was more potent than 5-FU and less toxic to human normal cells (IC 50 > 20 μM), indicating good selectivity. Specifically, by241 exhibited excellent inhibition against human gastric cancer cells (MGC-803 and BGC-803) with the IC 50 values of 2.77, 1.18 μM respectively. By241 inhibited growth of L-02 with an IC 50 value of 21.80 μM, showing around 5- and 8-fold selectivity toward SMMC-7721 (IC 50 = 4.83 μM) and ZIP77 (IC 50 = 2.71 μM), respectively. By241 also displayed similar selectivity to Het-1A (IC 50 = 20.15 μM) over other human esophageal cancer cells (EC109, EC9706 and KYSE450). For normal liver L-02 cells, by241 was about 5-fold less toxic than 5-FU, suggesting a relatively low toxicity of by241 (IC 50 = 21.80 μM vs. 4.37 μM). Additionally, a slight difference in inhibiting growth of sub-types of gastric cancer cells has also been observed (IC 50 = 2.77 and 1.18 μM, respectively against MGC-803 and BGC-803).

Table 1 In vitro cytotoxicity of by241 against several human cancer cell lines and two human normal cell lines. Full size table

After exposure to by241 at different concentrations for 72 h, the cell viability of cancerous cells and normal cells was decreased gradually with the concentration increase of by241, as demonstrated in Fig. 2A,B. For normal cells L-02 and Het-1A, the cell viability was almost unchanged even at high concentration (12.5 μM). In contrast, the viability of cancer cells dropped significantly, especially the BGC-803 and MGC-803 cells. The difference between normal cells and cancerous cells in cell viability also showed low toxicity and good selectivity of by241. At higher concentrations (>12.5 μM), the cell viability of normal cells declined substantially, suggesting the toxicity of by241 to normal cells at high concentrations.

Figure 2 Antiproliferative effects of by241 on selected human cell lines. (A) and (B) Human cells were treated with different concentrations of by241 for 72 h. Cell viability determined by MTT assay; (C) Representative images of MGC-803 cells colonies after treatment with various concentrations (0.31, 0.62 or 1.25 μM) of by241 for 12 days; (D) Quantitative analysis of the colony formation inhibition rate in Fig. 1C. Apoptosis related morphology and nuclear condensation of MGC-803 (E,F) induced by by241. Apoptosis related morphology and nuclear condensation of EC9706 (G,H) induced by by241. Cells were cultured with different concentrations of by241 (2.5, 5 and 10 μM) for 24 h and detected using the Hoechst 33258 assay. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 was considered statistically compared to control. Full size image

The clonogenic assay represents an indirect estimation of neoplastic transformation. As shown in Fig. 2C, smaller and fewer colonies were formed when MGC-803 cells were treated with increasing concentration of by241, the inhibition rate of colony formation was about 60% when treated with by241 at 1.25 μM for 12 days (Fig. 2D). Besides, morphological changes of MGC-803 and EC9706 cells such as rounding up and cell debris were observed, especially at high concentrations, after being incubated with by241 for 24 h at different concentrations (0, 2.5, 5, 10 μM) (Fig. 2E,G). After staining with Hoechst 33258, remarkable nuclear changes of MGC-803 and EC9706 cells including the chromatin condensation, nuclear fragmentation and condensation were also observed (Fig. 2F,H).

Cell apoptosis induced by by241

Above studies showed that by241 potently inhibited growth of cancer cells and induced morphological changes of MGC-803 and EC9706 cells in a concentration-dependent manner. These studies suggest that by241 should be capable of inducing apoptosis of cancer cells. Therefore, we performed the flow cytometric analysis of MGC-803 and EC9706 cells using the Annexin V-FITC and propidium iodide (PI) double staining after being incubated with by241 at different concentrations (0, 2.5, 5, 10 μM) for 12 or 24 h. As shown in Fig. 3A–D, by241 markedly induced apoptosis of MGC-803 cells in a concentration-/time-dependent manner, the late apoptosis in particular. Specifically, after treatment with by241 for 12 h, the apoptotic cells accounted for 28.3% at 10 μM, higher than that of the control group (Fig. 3A,C). More evidently, for the 24 h group, the apoptotic cells amounted to 85.8%, significantly higher than that of the control group (Fig. 3B,D). Besides, by241 also induced apoptosis of EC9706 cells in a concentration-/time-dependent manner (Fig. 3E–H). After treatment of EC9706 cells for 12 h at 10 μM, the percentage of apoptotic cells was 40.1%, significantly higher than the control group, but slightly lower than that of the group treated for 24 h at the same concentration (Fig. 3F,H).

Figure 3 By241 induced cell apoptosis. Apoptosis cells were detected using the Annexin V-FITC/PI double staining after exposure to by241 at different concentrations (0, 2.5, 5, 10 μM) and analyzed by flow cytometry and the apoptotic cell rate was analyzed after Annexin V-FITC/PI staining. (A,B) MGC-803 were treated for 12 h or 24 h; (E,F) EC9706 were treated for 12 h or 24 h; (C,D) Statistical analysis of apoptotic cells of (A,B); (G,H) Statistical analysis of apoptotic cells of (E,F); data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 were considered statistically compared to control. Full size image

By241 induced cell death through ROS-mediated mechanisms

The favorable potency of by241 toward cancer cells promoted us to investigate the potential mechanisms of action. More recently, Banerjee et al. designed a novel fluorescent cancer cell detector (named Is-Bet A) by combining the bis-arylidene oxindole and natural betulinic acid through an amino propyl-linker, in which betulinic acid, structurally belonging to steroids, acted as the ROS-generator, while the bis-arylidene oxindole served as a fluorophore for detection, thus achieving simultaneous detection and killing of cancer cells27. Mounting evidence has showed that steroid hormones can increase the ROS production in mitochondria28,29,30,31,32. Among them, DHEA has been proved to be able to increase ROS formation by inhibiting segment I of the respiratory chain33. Oliveira and co-workers reported that Uncaria tomentosa extract containing oxindole alkaloids triggered apoptosis of HT29 cells through ROS-mediated caspase activation and DNA repair34. Above studies suggest that both steroids and oxindole containing compounds could serve as ROS-generating agents for cancer therapy. From the structural point of view, we speculate that our target molecule by241 incorporating oxindole and steroid nucleus potentially induced cell death through ROS-mediated mechanisms.

As shown in Fig. 2E–H, by241 induced remarkable morphological changes of MGC-803 and EC9706 cells, especially at high concentrations. Interestingly, treatment of MGC-803 cells with N-acetyl-L-cysteine (NAC, 5 mM) rescued the by241-induced morphological changes of MGC-803 cells (Fig. 4A), suggesting that by241 probably induced cell death through elevating cellular ROS levels. We then used the DCFH-DA assay to determine the ROS levels in MGC-803 cells, after treatment with by241 at different concentrations (0, 5, 10 μM), following DCFH-DA treatment for 30 min, the green fluorescence was analyzed using an inverted fluorescence microscope. As shown in Fig. 4B, By241 concentration-dependently enhanced the green fluorescence intensity, the percentage of cells with green fluorescence amounted to 41.0% when treated at 10 μM, significantly higher than that of the control group (Fig. 4C) and NAC can decrease the green fluorescence intensity in MGC-803 cells induced by by241, the percentage of cells with green fluorescence was only 14.0% when treated with by241 (10 μM) and NAC (5 mM), slightly higher than that of the control group (8.8%). For EC9706 cells, the percentage of cells with green fluorescence intensity amounted to 35% (around 9-fold increase relative to the control) when treated with by241 at 10 μM, which then decreased to 9.2% when treated with by241 (10 μM) and NAC (5 mM) (Fig. 4E,F).

Figure 4 The role of ROS in by241-induced cancer cells death. After pretreatment with or without 5 mM NAC for 2 hours, MGC-803 cells were treated with or without by241 for 24 hours, then the apoptosis related morphology (A) and the intracellular ROS was detected by fluorescence microscope (B); After pretreatment with or without 5 mM NAC for 2 hours, MGC-803 cells (C) or EC9706 cells (E) were treated with or without by241 for 24 hours, then the intracellular ROS was detected by flow cytometry; (D,F) Statistical analysis of ROS levels of (C,E); (G) After pretreatment with or without 2 mM or 5 mM NAC for 2 hours, MGC-803 cells were treated with or without by241(6.25, 12.5 μM) for 24 hours, cell viability were detected by the MTT assay. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 and ##P < 0.01 were considered statistically compared to corresponding control. *P < 0.05, **P < 0.01 compared to control; ##P < 0.01 NAC+ by241-5 μM compared to by241–5 μM or NAC+ by241–10 μM compared to by241–10 μM. Full size image

NAC has been proved to be able to attenuate ROS generator-induced cancer cell death35,36. To investigate whether NAC can attenuate by241-induced MGC-803 cell death, NAC alone and in combination with by241 were used to explore the cytoprotective effect of NAC against the by241-induced MGC-803 cell death. As shown in Fig. 4G, NAC alone had no effect on the cell viability of MGC-803 cells even at the concentration of 5 mM, while NAC was found to be able to completely restore the decreased cell viability of MGC-803 cells caused by by241 at 6.25 μM. Around 80% of cell viability was observed when MGC-803 cells were treated with 12.5 μM and 5 mM of NAC (Fig. 4G). This result supported the hypothesis that by241 induced cell death mainly through ROS-mediated mechanisms.

To date, the relationship between mitochondrial damage and ROS changes is not clearly understood. It has been widely accepted that mitochondrial dysfunction is always associated with increased ROS production, accompanied with changes of mitochondrial membrane permeability, resulting in the loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of downstream caspases35. Hence, in this study, the ΔΨm was measured using the JC-1 dye to evaluate the by241-induced mitochondrial dysfunction (Fig. 5). As shown in Fig. 5A,B, by241 concentration-dependently increased the green fluorescence intensity in MGC-803 cells and EC9706 cells, indicating the by241-induced translocation of JC-1 dye from the mitochondria to the cytoplasm. The changes of the green/red fluorescence intensity in MGC-803 and EC9706 cells were then quantitatively analyzed using the flow cytometry analysis. As shown in Fig. 5C,D, a concentration-dependent decrease of MMP represented by the green/red fluorescence intensity was observed. At 10 μM, the fold of green/red fluorescence intensity of MGC-803 cells with the green fluorescence increased 12.0 fold. Similarly, the fold of green/red fluorescence intensity labeled EC9706 cells amounted increased 3.9 fold when treated with by241 at 10 μM, showing a concentration-dependent increase (Fig. 5E,F). Furthermore, the decreased MMP caused by by241 in MGC-803 could mostly reversed by NAC (Fig. 5G,H).

Figure 5 By241 induced mitochondrial dysfunction. Cells were treated with 5, 10 μM of by241 for 24 hours, JC-1 staining image of (A) MGC-803 and (B) EC9706 cells were detected by fluorescence microscope, JC-1 staining red and green fluorescence intensity of (C) MGC-803 and (E) EC9706 cells analyzed by flow cytometry. (D,F) Quantitative analysis of the ratio of green/red fluorescence in Fig. 5C,E. (G) MGC-803 Cells were pretreated with or without 5 mM NAC for 2 hours and then incubated with the by241 for another 24 hours, JC-1 staining red and green fluorescence intensity was analyzed by flow cytometry. (H) Quantitative analysis of the ratio of green to red fluorescence in Fig. 5G. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 were considered statistically significant compared with the controls, ##P < 0.01 NAC+by241-10μM compared to by241–10 μM. Full size image

Next, we examined the expression levels of mitochondria related Bcl-2 family proteins including pro-apoptotic proteins (Bax, Bid and Bak) and anti-apoptotic proteins (Bcl-2, Mcl-1 and Bcl-X L ). As shown in Fig. 6, after treatment with by241, expression levels of pro-apoptotic proteins increased concentration-dependently (Fig. 6E–G), particularly the Bid and Bax. Activated Bid is believed to be able to interact with Bax and then promotes the insertion of Bax into the mitochondrial outer membrane37. Besides, Bax and Bak are also able to promote the release of cytochrome c and other pro-apoptotic factors from the mitochondria, ultimately leading to activation of caspases by inducing the opening of mitochondrial voltage-dependent anion channel (VDAC) and/or forming the oligomeric pore MAC. Expressions of the anti-apoptotic proteins Bcl-2, Mcl-1 and Bcl-X L decreased correspondingly (Fig. 6B–D).

Figure 6 Expression changes of apoptosis related proteins induced by by241. (A) By241 induced expression changes of Bcl-2 family (Bcl-2, Mcl-1, Bcl-XL, Bak, Bid, Bax) proteins; (B–G) Statistical analysis of Bcl-2 family (Bcl-2, Mcl-1, Bcl-XL, Bak, Bid, Bax) proteins expression change induced by by241; (H) By241 induced caspase-3 and caspase-9 activation; (I–L) Statistical analysis of pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 expression levels; (M–P) By241 did not change the expression of TRL, DR5 and cleaved caspase-8. (Q) Effect of pan caspase inhibitor Z-VAD-FMK significantly attenuated by241-induced apoptosis MGC-803 cells; (R) Statistically analysis of the FITC-positive cells. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared to control; ##P < 0.01 Z-VAD-FMK+by241-5 μM compared to by241-5 μM. Full size image

Three major apoptosis-associated pathways to caspase activation have been identified and ordering of caspases involved is currently relatively well understood38,39. Above studies have shown that by241 markedly increased expression of pro-apoptotic proteins Bid, Bax and Bak, which have been proved to be able to activate expressions of downstream caspases. Herein, expression levels of three main caspases, namely the caspase-8, caspase-9 and caspase-3, were measured using the Western blot analysis. As shown in Fig. 6H, by241 concentration-dependently resulted in activation of pro-caspases-9 and -3, leading to increased expression of cleaved caspase-9 and caspase-3. Several reports described that ROS was capable of inducing cell death through activating caspase-840,41,42. However, in this study, the expression of cleaved caspase-8, DR5 and TLR was almost unchanged. Associated with overexpressed Bid (as shown in Fig. 6M), we reason that by241 induced apoptosis of MGC-803 cells in part through the mitochondria-related caspase-9/caspase-3 intrinsic pathways, not the membrane death receptor-mediated extrinsic pathways or the caspase-8/Bid/Bax pathway.

Furthermore, as shown in Fig. 6Q,R, pretreatment of MGC-803 cells with pan caspase inhibitor Z-VAD-FMK led to a decrease of FITC-positive cells (Apoptotic cells induced by by241). Specifically, the apoptotic cells for the by241-treated group accounted for about 22%, while the percentage of apoptotic cells after treatment with by241 and Z-VAD-FMK decreased to around 7%, but still slightly higher than that of cells treated with Z-VAD-FMK alone (about 5%, Fig. 7R). These results revealed that pan caspase inhibitor Z-VAD-FMK cannot prevent by241-induced apoptosis completely, suggesting that apart from the mitochondria-mediated apoptotic pathways, simulation of other signaling pathways and/or key proteins involved in the ROS-mediated pathways by by241 also contributed to MGC-803 cell death.

Figure 7 Expression changes of key proteins involved in ROS mediated pathways. (A) Expression analysis of p-NF-κB, MDM2, p53, p21 and PUMA in by241-treated MGC-803 cells. Western blot of protein extracted from MGC-803 cells following 24 h treatment with by241 (5 μM and 10 μM), a representative result of 3 independent experiments is shown. (B–F) Statistical analysis of p-NF-κB, MDM2, p53, p21 and PUMA proteins expression change induced by by241; (G) Expression analysis of p-PI3K, p-AKT (Thr308), p-AKT (Ser473), AKT, p-mTOR (Ser2448) and mTOR in by241-treated EC109 cells. (H–K) Statistical analysis of p-PI3K, p-AKT (Thr308), p-AKT (Ser473) and p-mTOR (Ser2448) proteins expression changes induced by by241; Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 were considered statistically significant compared with the controls. Full size image

Apart from the mitochondria-mediated apoptotic pathways, mounting evidence has shown that ROS can regulate many other signaling pathways such as p5343,44,45,46,47. Activated p53 then induces expression of downstream transcriptional proapoptotic proteins such as Bax and PUMA48, ultimately leading to apoptosis of cancer cells. We next examined expression changes of key proteins related to ROS-mediated apoptosis induced by by241. As shown in Fig. 7, treatment with by241 increased expression of p53, which then transcriptionally activated its downstream proapoptotic proteins such as Bax (Fig. 6A) and PUMA (Fig. 7A). p53 also induced expression of p21 (Fig. 7A), which, as the cyclin-dependent kinase (CDK) inhibitor, can arrest cell cycle at G1 phase49,50 and is also able to inhibit cancer cell growth51,52. Also, by241 concentration-dependently cleaved MDM2, generating a 60KD MDM2 fragment (Fig. 7A), which at least in part could be explained by the caspase-3 activation. It has been reported that caspase-3 can cleave the MDM2 oncoprotein during p53-mediated apoptosis53,54.

The transcription factor NF-κB plays vital roles in regulating cell differentiation, responses to oxidative stress and apoptosis through controlling expression of related genes55. The crosstalk between NF-κB and p53 is regulated by the relative levels of each transcriptional factor. ROS-induced p53 activation therefore could down-regulate NF-κB, accompanied with increased expression of phosphorylated NF-ĸB (p-NF-ĸB) (Fig. 7A). To date, a large number of natural and synthetic small molecules, as well as peptides have been identified as NF-κB inhibitors56. However, the therapeutical effect of NF-κB inhibitors as anticancer agents is controversial. Recently, Ryan et al. described that inhibition of NF-κB in wild-type p53 retaining tumors may cause a diminished therapeutical response57.

Besides, ROS also activated the PI3K/AKT pathways58, leading to increased expression of key proteins such as PI3K and AKT (Fig. 7G). mTOR, as a conserved serine/threonine kinase, is crucial in regulating cell survival and proliferation. As shown in Fig. 7G, the expression of p-m-TOR was down-regulated, which could be achieved through the ROS-JNK-p53 pathway47 or by activating AMPKα59.

In vivo acute oral toxicity and anti-tumor activity of by241

Due to the favorable potency in inhibiting growth of cancer cells in vitro and low toxicity to normal cells, we then evaluated the in vivo acute oral toxicity of by241 on mice, which may provide a guideline for selecting doses for further in vivo experiments. As shown in Table 2, no severe side effects or mortality in test groups were observed even at the dose of 1000 mg/Kg. Animals did not show significant abnormal signs, behavioral changes and water or food consumption during observation. As shown in Fig. 8A, body weight changes of animals treated with by241 relative to the control was not remarkable. Furthermore, no significant changes or lesion in the viscera of test animals were observed in autopsy experiments.

Table 2 The acute oral toxicity of by241 at fixed doses. Full size table

Figure 8 The acute oral toxicity of by241 on mice and antitumor efficacy in MGC-803 bearing nude model. (A) Body weight of mice in 15 days after oral treatment with by241 (250, 500 or 1000 mg/kg). MGC-803 cells were transplanted subcutaneously to the BALB-C nude mice and subjected to by241 (40, 80 and 120 mg/kg), 5-FU (15 mg/kg) and saline were used as the negative control for 21 days. (B) Tumor size and (C) body weight measurements every 2 days from MGC-803 mice after by241 administration. (D) Photographs of mice in saline group, 5-FU group and by241 (120 mg/kg) group and (E) Photographs of tumors in each group. (F) Comparison of the final tumor weight in each group after 21-day treatment. Data are presented as means ± SD. **P < 0.01 was considered statistically significant compared with the negative control. Full size image

The favorable in vitro potency and low toxicity of by241 observed promoted us to investigate the in vivo anti-tumor potency. MGC-803 cells were injected in the paw subcutaneous of the mice. Three days later, by241 were intragastrically administered to the mice daily at different doses (40, 80 and 120 mg/Kg) using 5-FU (15 mg/Kg), saline as the positive and negative controls, respectively. During the 21-day treatment, the mouse body weight was monitored every 2 days. As shown in Fig. 8C, compared to the controls, the mice administered daily with by241 even at 120 mg/Kg did not show any body weight loss, while the body weight of mice treated with 15 mg/Kg 5-FU daily declined gradually during the last 4-day treatment, showing less toxicity of by241 than 5-FU after repeated 21-day treatment at high doses. Besides, as shown in Fig. 8B, by241 exhibited favorable anticancer efficacy at 40 mg, 80 and 120 mg/kg, the corresponding tumor volume for the by241 (120 mg/kg) treatment group was significantly smaller than that of the saline group. At the end of the experiment, tumor volume was 2126 mm3 in the control group, while tumor volume in the treatment groups was 1247 mm3, 1060 mm3 and 957 mm3 at by241 doses of 40 mg/kg, 80 mg/kg and 120 mg/kg, respectively. The tumor volume of the 5-FU group was 822 mm3. Compared to the control group treated with saline (1.3 g), the average tumor weight of mice treated with by241 at 40, 80 and 120 mg/kg decreased to around 0.87 g, 0.67 g and 0.52 g, respectively (Fig. 8E,F), accounting for a 32.3%, 47.7% and 59.6% reduction in tumor weight. And the average tumor weight of the 5-FU group was 0.51 g, showing 59.8% decrease in tumor weight. The favorable in vitro and in vivo anticancer potency and the absence of acute oral toxicity even at high doses (Table 2 and Fig. 8A) warrant its further development for cancer therapy. As shown in Table 1, by241 was more potent than 5-FU against tested cancer cells, but the inferior in vivo anticancer activity of by241 could be attributed to the relatively poor bioavailability of by241.