Background
Colon cancer is a frequent occurrence in malignancies of digestive tract with an increasing mortality rate, but a low 5-year survival rate [
1]. Many significant therapeutic agents over the past decade have been applied for colorectal cancer therapy [
2,
3]. However, chemotherapeutic agents gradually turn out to show its drawbacks due to lack of specificity or drug resistances [
4,
5]. In this regard, potential drugs with specific target are now being developed for clinical application to cure colorectal cancer patients.
SC66, a novel AKT inhibitor, has shown greater promise than other PIP3/Akt inhibitors against several cancer types, including cervical cancer [
6]. As an allosteric AKT inhibitor, SC66 facilitates Akt deactivation more effectively by directly interfering with the PH domain binding to PIP3, and subsequently induces Akt ubiquitination, thereby manifesting a more efficient growth suppression of transformed cells that are associated with a high-level expression of Akt signaling [
7]. SC66 also induces alterations in cytoskeleton organization and ROS production, leading to a reduction in total and phospho-AKT levels. SC66 has been demonstrated to inhibit tumor growth of hepatocellular carcinoma significantly via the AKT/mTOR/β-catenin pathway [
8]. However, the mechanisms by which SC66 exerts its antitumor activity especially how to induce cell apoptosis are not well-understood.
One of the most typically activated pathways in human colon cancer is the PI3K/AKT signaling, which has been involved in tumor initiation, invasion, vascularization and metastasis [
9‐
12]. It is therefore not surprising that this inappropriately activated signaling contributes to making the AKT important therapeutic target. AKT, also known as protein kinase B (PKB), is a serine/threonine kinase that mediates cell proliferation, protein synthesis, transcription, and apoptosis [
13‐
15]. And its kinase activity is positively mediated by phosphorylation on two key residues Ser473 and Thr308 [
16]. Phosphorylation of both residues on is required for maximal AKT1 activation downstream of PI3K [
17]. Once activated by a variety of apoptotic stimuli, AKT inhibits the function of the critical tumor suppressor p53 and promotes survival [
18]. In addition, several myriad substrates, such as Forkhead Box O3a (FoxO3a), nuclear factor κB (NF-κB) and the mammalian target of rapamycin (mTOR), may be activated via the PI3K/Akt axis [
11,
19,
20]. Akt phosphorylates and inhibits several pro-apoptotic gene activities such as Bad, Bim and procaspase 9 [
10,
15,
21,
22]. Importantly, activated AKT protects cells from pro-apoptotic stimuli as well as inhibiting GSK-3β [
23].
Glycogen synthase kinase-3β (GSK-3β) is a key mediator of apoptosis responding to numerous stimuli [
24,
25]. Current studies have demonstrated that active GSK-3β promotes the mitochondrial localization of Bax and induces neuronal apoptosis in response to staurosporine or heat shock [
26,
27]. Several Bcl-2 family members are direct substrates/indirect targets of GSK-3β [
13]. For example, activated GSK-3β phosphorylates the transcription factor c-Myb, leading to the altered level of Bcl-2 [
28,
29]. Pro-apoptotic BH3-only members such as PUMA, Bim, and Bid, indirectly or directly activate the multi-BH domain containing pro-apoptotic signals Bax/Bak, which triggers Bax oligomerization and subsequently causes downstream events including mitochondrial outer membrane permeabilization (MOMP), caspase cascade, and apoptosis [
30‐
33]. Bcl-xL, a critical pro-survival Bcl-2 family member, suppresses apoptosis through the hydrophobic BH3 domain-binding groove of pro-apoptotic signals [
34]. Several studies reported certain solid tumor cell lines with low Bcl-xL were sensitive to conventional therapies.
Materials and methods
Cell culture and treatment
The human colon cancer cell lines, HCT-116, RKO and DLD1 were obtained from American type culture collection (ATCC). Human colon cancer cell line with p53−/− (HCT-116 p53-KO), and Bax−/− (HCT-116 Bax-KO) were generously provided by Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD, USA). All the cell lines were routinely cultured in McCoy’s 5A modified medium, supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 mg/ml) in 5% CO2 at 37 °C in humidified incubator. SC66 was purchased from Selleck Chemicals (Houston, TX). The agent of SC66 diluted with DMSO were added in the medium directly. For treatment, the anticancer agent was added in the medium directly before detection.
Transfections were performed with Lipofectamine™ 2000 reagent according to the manufacturer’s protocols (7 sea biotech, Shanghai, China). Cells were transfected with either an empty vector or a constitutively-active Akt expression constructs for 48 h. Then added the related drugs into the culture medium, and replaced with fresh culture medium after 6 h and kept for 12 h. Finally, cells were harvested and proteins were examined to at 24–48 h after transfection.
Antibodies and reagents
Primary antibodies against p53, Phospho-Akt (S473), Total-Akt, Phospho-GSK-3β, GSK-3β, Bax, Bcl-2, Bcl-XL, Mcl-1, Cox IV, Cleaved-Caspase3 and β-actin were purchased from Cell Signaling Technology. Lipofectamine™ Reagent was purchased from Invitrogen. HRP-conjugated anti-rabbit and or anti-mouse secondary antibodies an ECL-plus kit were from GE Healthcare. SC66 was purchased from Selleck Chemicals (Houston, TX). The oligonucleotide for shGSK-3 was synthesized as 5′-CCGGGTGTGGATCAGTTGGTAGAAACTCGAGTTTCTACCAACTGATCCACACTTTTT-3′. The CCK-8 kit was from 7 sea biotech (Shanghai, China).
Cell viability and apoptosis assays
Colon cancer cells were cultured in 96-well microplate at a density of 3.5 × 103 cells/well for 24 h. Cell viability was assessed with CCK-8 at indicated time post treatment according to the manufacturer’s instructions. To estimate the viability of the cells, the absorbance of 450 nm (OD450) was measured with a 96-well plate reader (DG5032, Huadong, Nanjing, China).
For analysis of apoptosis by Hoechst 33342 (Invitrogen), colon cells were cultured on the coverslip of a chamber, rinsed with phosphate-buffered saline (PBS) twice, and then added in 1 ml of McCoy’s 5A containing 1 μl Hoechst 33342, incubated at 37 °C with 5% CO2 for 15 or 20 min. Apoptosis was assessed through microscopic visualization of condensed chromatin and micronucleation.
For colony formation assays, equal number of cells after different treatments were plated into 6-well plates. Medium was changed every 2 days. Colonies were visualized by crystal violet staining 2 weeks after plat.
Western blotting
Protein samples were extracted with RIPA buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS. 140 mM NaCl). Equivalent protein samples (30 μg protein extract was loaded on each lane) were subjected to SDS-PAGE on 10% gel or 12% gel. The proteins were then transferred onto PVDF membranes (Millipore) and blocked with 5% non-fat milk for 1 h at room temperature. The membranes, probed with the indicated primary antibodies, were then incubated at 4 °C overnight. Primary antibody was detected by binding horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody with an ECL plus kit. Detection was performed using the Odyssey infrared imaging system (LI-COR, Lincoln, NE). To detect Bax multimerization, purified mitochondrial fractions were cross-linked with DSP [dithiobis (succinimidyl propionate)] (1 mmol/l), followed by Western blotting analysis.
Flow cytometry
After treatment, human colon cancer cell line with HCT-116 WT, p53−/− (HCT-116 p53-KO), DLD1 and Bax−/− (HCT116 Bax-KO) were suspended in 1 × 105 cells/ml, and 5 μl Annexin V and 5 μl propidium iodide staining solution were added to 100 μl of the cell suspension. Then added 400 μl Binding Buffer to the cell suspension again. After the cells were incubated at room temperature for 10 min in the dark, stained cells were assayed and quantified using a FACSort Flow Cytometer (Beckman Coulter, Brea, CA, USA). Cell debris was excluded from the analysis by an appropriate forward light scatter threshold setting. Compensation was used wherever necessary.
Co-immunoprecipitation
To detect the interaction between GSK-3β and Bax, about 4 ml of GSK-3β or Bax antibodies respectively were firstly added to 400 ml cell lysates. According to the manufacturer’s protocol, the mixtures were mixed on a rocker at ambient temperature for 2 h. The immunocomplexes were captured by the addition of protein A-agarose (Roche Applied Sciences, Indianpolis, Cat. No. 11 134 515 001) mixed at 1:10 ratio, followed by incubation at ambient temperature for 1 h. The beads were washed by Washing Buffer 1, Washing Buffer 2, Washing Buffer 3 (Roche Applied Sciences, Indianpolis, Cat. No. 11 134 515 001) and then collected by centrifugation at 12,000 rpm for 30 s. After the final wash, the beads were mixed with 60 ml of 2× Laemmli sample buffer, heated at 98 °C for 8 min, and analyzed by Western blotting using GSK-3β, Bax6A7 or Bax antibody (Cell Signaling Technology, Shanghai, China).
Xenograft mouse model and treatment
Female 5-week-old nude mice (Vital River, China) were housed in a sterile environment with micro isolator cages and allowed access to water and chow ad libitum. HCT-116 WT was harvested, and 1 × 106 cells in 0.2 ml of McCoy’s 5A modified medium were implanted subcutaneously into the back of athymic nude female mice. Mice were treated with daily with SC66 at 25 mg/kg by i.p. injection every other 3 days for 15 days, whereas the control mice were administered vehicle. Volume was calculated by the formula of 0.5 × length × width2. Mice were euthanized when tumors reached ~ 1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. All mice were housed and maintained under specific pathogen-free (SOPF) conditions. All animal studies were in accordance with institutional guidelines and approved by the Use Committee for Animal Care.
Statistical analysis
Statistical analyses were performed using GraphPad Prism V software. All assays were repeated independently more than three times. Data are represented as mean ± SEM in the figures. P values were calculated using the Student’s paired t-test.
Discussion
Colorectal cancer remains the third most commonly diagnosed malignancies in the worldwide, which has been estimated about 1.2 million new cases and almost 600,000 deaths annually [
36,
37]. Resistance to conventional systemic frontline therapies is a big hurdle in treating colon cancer and reducing serious side-effects due to lack of specificity such as 5-FU, leucovorin, oxaliplatin and irinotecan [
38‐
40]. As we all know, the aberrant activation of AKT/PKB pathway in a variety of cancers including colon cancer is crucial in the proliferation, resistance to apoptosis, metabolism and angiogenesis. AKT mediates cell survival and death by directly and indirectly phosphorylating pro-apoptotic Bcl-2 family proteins or regulating transcriptional factors (for example, FoxO3a, NF-κB, p73 and p53) [
15,
19,
41‐
43]. Therefore, novel therapeutic strategies are needed to advance for cancer patients, suggesting designing of new drugs targeting alternative AKT signaling may be more attractive and appeal for therapeutic intervention.
Historically, several available molecules inhibiting AKT are categorized into three groups including ATP-competitive inhibitors, phosphatidylinositol analogs, and allosteric inhibitors [
17]. Novel agents such as NVP-BEZ235 and MK-2206, are now in preclinical development [
44‐
47]. More recently, SC66 is a potent and highly selective allosteric pan AKT inhibitor, which facilitates AKT ubiquitination and further inhibits glucose uptake in vitro and in vivo. Recent studies have reported that SC66 has shown a strong anti-tumor activity, including hepatocellular carcinoma and cervical cancer with PIK3CA R88Q and PTEN R233* mutation [
6,
8]. However, the antitumor effects of SC66 and the underlying mechanism in colon cancer still remain to be elucidated.
In the current study, we investigated the potential antitumor activity of SC66 on colon cancer cell lines, showing that colon cancer growth was effectively suppressed after SC66 simulation (Figs.
1,
2,
5 and
6). Next, we further studied the precise mechanism of SC66 induced-apoptosis. Using multiple cell lines including WT, p53 mutant or knockout cells, we demonstrated that SC66 induced apoptosis through p53-independent pathway for the first time (Figs.
1,
2 and
3).
Previous reports from our laboratory have demonstrated that low-power laser irradiation exerts protective effects in preventing cell apoptosis by activating AKT and inactivating GSK-3β [
35]. Current studies have demonstrated oligo-porphyran offers a neuroprotective treatment for Parkinson’s disease via PI3K/Akt/GSK-3β pathway, with changes in the Bax/Bcl-2 ratio [
48]. However, some studies showed that activated GSK-3β promoted Bax activation in a p53-dependent pathway [
49]. Our results showed that AKT activity was highly inhibited in various colon cancer cell lines after SC66 treatment, suggesting AKT signal is independent of p53. The canonical pathway of GSK-3β activation is mediated by NF-κB phosphorylation, for example, in response to sorafenib [
42]. Notably, we confirmed SC66 showed greatly suppression of AKT activity and induced apoptosis through the significant factor GSK-3β, but not depend on the important factor such as p53 (Figs.
2,
3).
AKT inhibition led to activation of GSK-3β, that promoted Bax translocation by directly binding to Bax, simultaneously decreasing Bcl-xL, and subsequently triggering the mitochondrial apoptosis (Figs.
3,
4,
5). In addition, GSK-3β/Bax axis is indispensable for using SC66 to treat colon cancer (Figs.
4,
5,
6). The influence of Bax deficiency on SC66 induced-apoptosis is inferior to that of GSK-3β deletion in HCT-116. These results above indicated that SC66 regulated cell survival and apoptosis via the AKT/GSK-3β/Bax pathway, which is distinct from previous reports involved the PI3K/Akt/mTOR or PI3K/PKB/Egr-1 pathway [
6,
50].
Research has identified that SC66 interferes with the PH domain binding to PIP3 and directly enhances Akt ubiquitination and its deactivation in both HeLa and HEK293T cells [
7]. Recent report claims that SC66 had significant anti-tumor effects on hepatocellular carcinoma cells through producing ROS, subsequently inducing anoikis-mediated cell death and inhibiting the AKT signaling pathway [
8]. And the novel Akt inhibitor SC66 directly deactivated Akt and facilitated the activation of Foxo, eventually triggering apoptosis. Some studies also indicated that SC66 effectively inhibited the phosphorylation levels of AKT through disruption of mTOR signaling, and therefore contributes to decrease the expressions of p-GSK-3β and p-FOXO1 in human cervical cancer [
6]. Further study in this area may be instructive in different Akt inhibitors, dual PI3K/mTOR inhibitors or other multi-kinase inhibitors.
Previous studies also demonstrated Akt inhibition by pazopanib, ipatasertib or NVP-BEZ235 induced PUMA-dependent apoptosis in colon cancer through activating FoxO3a transcriptionally [
32,
33,
51]. Besides, the results indicated that Mcl may also play some roles while Bax and Bcl-xL seem to be the key regulators of mediating cell apoptosis (Fig.
3). A recent study indicated that regorafenib induced colon cancer cell apoptosis via GSK-3β/Mcl-1 axis [
52]. Therefore, it seems a link between GSK-3β and Mcl is complicated, which is worth to be further studied.
In conclusion, we provided the first evidence that SC66 exerts its antitumor effects through GSK-3β/Bax pathway through AKT inhibition and initiates apoptosis through the mitochondrial pathway, which is p53-independent. GSK-3β deletion or Bax deficiency abrogated SC66-induced apoptosis and promoted colon cell survival. Together with the data from xenograft mice in vivo, we highlight the novel AKT inhibitor SC66 may function as a promising and potential therapeutic drug for colon cancer treatment, providing the rationale for clinical application in the future.
Authors’ contributions
YL: study conception and data acquisition and interpretation and draft the manuscript; YH, JD and NL: analyses and collection; SP: data acquisition and interpretation; JW, FW and YZ: study design and data supervision and contributed to manuscript review. All authors read and approved the final manuscript.