Pyrrolo [3,4-b]-quinolin-9-amine compound FZU-0038-056 suppresses triple-negative breast cancer partially through inhibiting the expression of Bcl-2

Triple-negative breast cancer (TNBC) has a poorer prognosis than other subtypes of breast cancer; however, it lacks effective targeted therapies clinically. In this study, we found FZU-0038-056, a novel compound derived from last-stage functionalization of tetrahydro-β-carboline scaffold, showed the most potent anti-cancer activity against TNBC cells among the 42 synthesized derivatives. We found FZU-0038-056 significantly induces apoptosis in HCC1806 and HCC1937 TNBC cells. FZU-0038-056 reduces the expression levels of several anti-apoptosis proteins, including Bcl-2, Mcl-1 and XIAP. Furthermore, we found FZU-0038-056 induces apoptosis partially through inhibiting the expression of Bcl-2. Finally, we found FZU-0038-056 significantly suppresses HCC1806 xenograft tumor growth in nude mice without affecting their body weight. Therefore, FZU-0038-056 has the potential to be a new anticancer agent for treating human TNBC.


INTRODUCTION
Breast cancer ranks first among female malignant tumors [1]. According to the cancer statistics of China, the incidence of breast cancer in China is increasing at a rate of 3-4 % per year, and the age of onset is also gradually becoming younger [2]. Based on gene expression profiles, breast cancer can be divided into four subtypes, luminal A, luminal B, HER2 positive, and basallike/triple-negative breast cancers (TNBC) [3,4]. Among all the subtypes of breast cancer, TNBC is more aggressive and has higher rates of relapse and metastasis than other subtypes. In the past decades, hormone therapy and anti-HER2 targeted therapy have significantly improved the prognosis of ERα/PR+ and HER2+ breast cancers, respectively. However, TNBC does not have effective targeted therapies for lacking the expression of ERα, PR and HER2, chemotherapy remains the major option for TNBC patients [5]. Therefore, it is important to develop effective treatments for TNBC.
Mitochondria are cellular organelles that engage in aerobic respiration. In addition to producing ATP, they are also involved in apoptosis. Mitochondria are the central organelle in the intrinsic apoptotic pathway, acting through the release of cytochrome C, Smac/DIABLO (direct IAP binding protein) and apoptosis-inducing factor AIF, while Bcl-2 can inhibit apoptosis by inhibiting the release of cytochrome C and preventing the translocation of Smac/DIABLO and the apoptosis-inducing factor AIF [6,7].
The tetrahydro-β-carboline (THβC) skeleton as one important class of natural indole alkaloids commonly was found in nature. It is a very important skeleton and is present in many pharmacologically active alkaloids such as reserpine, tadalafil, and tetrahydro-halamine [8]. In recent years, tetrahydro-β-carboline derivatives have also become popular in terms of cancer targeted therapy. The TGF-β signaling pathway [9], phosphodiesterase (PDE) [10,11], and spindle kinesin (KSP) [12,13] are potential anti-tumor targets of tetrahydro-βcarboline derivatives. To develop more potent anticancer reagents, we designed and synthesized 42 pyrrolo [3,4-b]-quinolin-9-amines by using THβC as the starting point through an oxidative rearrangement coupling reaction.
There are no reports on the anti-tumor activity of pyrrolo [3,4-b]-quinolin-9-amine compounds to date. In this study, we screened anti-cancer activities of the 42 compounds and identified FZU-0038-056 to be the most potent one. FZU-0038-056 significantly induced apoptosis in HCC1806 and HCC1937. We showed that FZU-0038-056 reduced the protein expression levels of Bcl-2, Mcl-1 and XIAP. Bcl-2 overexpression partially reduced the pro-apoptosis of FZU-0038-056. These findings suggest that FZU-0038-056 is a mitochondrial apoptotic pathway activator and may be an effective anticancer agent for the treatment of TNBC.

FZU-0038-056 regulates the expression of apoptosisrelated genes
Since FZU-0038-056 induced apoptosis in HCC1806 and HCC1937 cells, we further examined the expression of apoptosis related genes by WB. FZU-0038-056 treatment increased the cleavage of caspase-3 and PARP in the HCC1806 and HCC1937 cell lines ( Figure  3A). Furthermore, it significantly decreased the protein expression levels of several anti-apoptotic proteins, including Bcl-2, XIAP, and Mcl-1, in a dose-dependent manner ( Figure 3A). In contrast, we did not observe an increase of expression of pro-apoptosis proteins, including Bax, Bak, and DR5 ( Figure 3A).
In addition, we examined the activation of several major apoptosis-related signaling proteins, including p38, JNK, ERK, and AKT. We found FZU-0038-056 increased the phosphorylation level of p38, but not the other tested kinases, in HCC1806 and HCC1937 cells in a dose-dependent manner ( Figure 3B).

FZU-0038-056 does not induce TNBC apoptosis through activating p38
The p38 MAPK signaling pathway is well known to play important roles in various physiological processes, including apoptosis [14]. To test whether p38 activation leads to apoptosis, we knocked down p38 using two AGING different siRNAs in HCC1806 and HCC1937 cells ( Figure 4A, 4B). However, depletion of p38 did not attenuate the cell survival inhibition effects of FZU-0038-056 in either of the tested TNBC cell lines.
Although FZU-0038-056 could efficiently kill cancer cells in our study, the dosage used is relatively high in vitro ( Figure 1A, 1B) and in vivo ( Figure 6A, 6C). Considering the side effects of cisplatin is severe, while FZU-0038-056 is much safer in vivo ( Figure 6D), we tried to check whether FZU-0038-056 could strengthen the anti-tumor effect of cisplatin, thus cisplatin could be applied at lower dosages. As the data shown in
Apoptosis is one of the main mechanisms by which drugs inhibit tumor growth. There are three main pathways for apoptosis: the mitochondrial pathway, the death receptor pathway, and the endoplasmic reticulum pathway [16]. The mitochondrial programmed death pathway is a process in which multiple factors are interrelated and balance each other to promote cell death [17,18]. When the apoptotic signal is transduced into mitochondria, the mitochondrial outer membrane  forms poly channels, and mitochondrial contents such as cytochrome C (cytC), Smac and AIF are released [19]. Smac can relieve the inhibition of precursor caspase by IAPs. AIF induces fragmentation of DNA and concentration of chromosomes [19]. CytC can activate caspase-9 [20]. In this study, FZU-0038-056 downregulated the expression of Bcl-2 and Mcl-1 and induced the cleavage of caspase-3 and PARP (Figure 3). Meanwhile, the proapoptotic effect of FZU-0038-056 on TNBC cells can be significantly reduced by ectopically overexpressed Bcl-2 ( Figure 5). Therefore, FZU-0038-056 activates the intrinsic mitochondrial apoptosis pathway partially by inhibiting the expression of Bcl-2. However, the exact mechanism by which FZU-0038-056 inhibits the expression of Bcl-2 is still unclear and requires further investigation. It would be interesting to identify direct binding molecules of FZU-0038-056.
In summary, we demonstrated that FZU-0038-056 induces apoptosis of TNBC cells and inhibits TNBC tumor growth in nude mice. The mechanism by which FZU-0038-056 inhibits TNBC may involve the suppression of Bcl-2. Interestingly, we found combination application of FZU-0038-056 and cisplatin synergistically suppressed HCC1806 cell survival, which implicates possible application of FZU-0038-056 in combination with clinically used anti-tumor agents, including cisplatin. Therefore, FZU-0038-056 has potential as a novel anticancer agent for human TNBC.

Compounds and cell lines
FZU-0038-056 and other compounds were designed and synthesized from THβC as the lead compound [8]. FZU-0038-056 was dissolved in DMSO and diluted in the corresponding culture media for the experiments. All of the cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and validated by STR analysis (Kunming Cell Bank, Kunming Institute of Zoology, Chinese Academy of Sciences). HCC1806 and HCC1937 were cultured in RPMI-1640 medium supplemented with 10 % fetal bovin serum (FBS). MDA-MB-231 and MDA-MB-468 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10 % FBS. MCF7 was cultured in Minimum Essential Medium (MEM) with 10 % FBS and 0.01 mg/ml human recombinant insulin. 184B5 cells were cultured in DMEM/F12 with 10 % FBS, 5 µg/ml insulin and 10 ng/ml cholera toxin. All cells were maintained at 37 °C with 5 % CO2 in a humidified atmosphere.

In vitro cytotoxicity assays
Normally cultured logarithmic growth phase cells were uniformly seeded at 30,000/well into 48-well plates. The day after plating, the cells were treated with the drugs at the indicated concentrations. Forty-eight hours later, the cells were fixed with 200 µl 10 % TCA (trichloroacetic acid) solution for 1 hour at room temperature, washed 5 times with deionized water, and then dried at room temperature. After drying, the cells were stained with 100 µl of 0.4 % (W/V) SRB in 1 % acetic acid for 5-15 min, followed by washing 5 times with 1 % glacial acetic acid and dried. Finally, 200 µl of 10 mM Tris base solution per well was added, and optical densities were measured at 530 nm in a spectrophotometric plate reader.

Apoptosis analysis
HCC1806 and HCC1937 were treated with different concentrations of FZU-0038-056 for 24 hours. DMSO was used as the negative control. The cells were stained with Annexin-V (BD, San Diego, CA) for 30 min and PI (BD, San Diego, CA) for 5 min in the dark at room temperature. Finally, the cells were analyzed by flow cytometry.

Western blotting (WB)
FZU-0038-056 or DMSO treated cancer cells were harvested using lysis buffer supplemented with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 30 min on ice. Proteins were collected and centrifuged at 4 °C, 13000 rpm for 10 min. Equal amounts of protein samples were then separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membrane was blocked in 5 % skim milk for 1 hour, incubated with the primary antibody (1000 × dilution) overnight at 4 °C, washed 3 AGING times with 1 × PBST for 10 min/time, and then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (Jackson Immuno-Research Laboratory, West Grove, PA) for 1 hour. They were then washed 3 times again with 1 × PBST. Finally, the membranes were incubated with Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Shelton, CT) and images were taken using an ImageQuant LAS4000 Biomolecular imager (GE Healthcare, UK).

Tumorigenicity assays
Eighteen six-week-old female nude mice were purchased from Hunan SJA Laboratory Animal Co., Ltd., (Changsha, Hunan, China) and were housed in an SPF animal facility. HCC1806 cells (1×10 6 cells per mouse, resuspended in 75 μl 1×PBS with 20 % Matrigel) were injected subcutaneously near the third mammary fat pad of the mice. When the tumor grew to 50 mm 3 , the mice were randomly divided into 3 groups (n=6). All mice were injected intraperitoneally (i.p.) with FZU-0038-056 solution or control every two days. The negative control group was treated with vehicle solution (5 % DMSO + 95 % saline), the experimental group was treated with FZU-0038-056 (15 mg/kg), and the positive control group was treated with cisplatin (8 mg/kg). The body weights and tumor volumes were measured every 4 days and the tumor volumes were calculated according to the formula V = 0.5 × L × W 2 , where L = length (mm) and W = width (mm). Animal studies were approved by the Institutional Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences.

Statistical analysis
All data in this study were analyzed by SPSS 7.0 and are presented as the mean ± SD. Differences between groups were identified using Student's t-test, and p<0.05 was considered statistically significant.