The ubiquitin-proteasome system plays a pivotal role in maintaining the balance between normal growth and uncontrolled proliferation by controlling the abundance of a large variety of cellular proteins . The cullin family of ubiquitin ligases, traditionally composed of CUL1, 2, 3, 4A, 4B, 5 and 7, represents the largest class of RING-type E3 ligases (CRLs) . Without intrinsic catalytic activity, cullins serve as scaffolds that facilitate the assembly of multimeric E3 ligase complexes and transfer ubiquitin from the E2-conjugating enzyme to the substrate. Cullin-mediated substrate degradation dictates a wide range of cellular processes such as proliferation, differentiation, and apoptosis. Once the cell regulatory mechanisms of cullin encounter malfunctions or perturbations, accumulation of oncoproteins or excessive degradation of tumor suppressors will inevitably occur, which may provoke cells into malignant transformation and tumorigenesis . In particular, cullin1, the most characterized member of the cullin family, was proven to be closely associated with gastric carcinogenesis, and its overexpression predicts poor prognosis of patients with gastric carcinoma [6, 7].
The expression profiles of cancer-related genes in part reflect their biological features in the pathogenesis of cancer. Being a novel member of the cullin family, CAC1 expression patterns have been extensively investigated in a previous study . Using cDNA library analysis, CAC1 expression was found in normal stomach, small intestine, colon, liver, lung, kidney, muscle, heart, mammary gland, uterus, brain, spleen, lymph node, with high levels in the colon and mammary gland . Western blot tests additionally detected CAC1 protein in a host of normal and cancer cell lines . With regard to our study, the AGS and MGC803 gastric cancer cell lines had higher CAC1 expression than the GES-1 gastric mucosal cell line, which indicate that CAC1 may play an active role in gastric carcinogenesis.
Gene silencing by RNA interference is a powerful method for analyzing gene function . Here, we successfully transfected NCsiRNA and three concentrations of CAC1-siRNA into AGS cells. MTT analyses showed that the proliferation potential of AGS cells was potently inhibited by 60nM and 90nM CAC1-siRNA to the same degree, but failed to be influenced by 30nM CAC1-siRNA or NC-siRNA. It was apparent that CAC1 could positively affect cell proliferation in gastric cancer, which was in accord with previous studies on the HeLa cell line . In MTT examinations, HeLa cells expressing Flag-CAC1 underwent higher proliferation ability than the mock transfected control cells, and cells treated with CAC1-siRNA showed significantly inhibited growth rate . What’s more, real-time RT-PCR and western blot analyses confirmed that the expression of CAC1 was efficaciously blocked by 60nM CAC1-siRNA. Taken together, 60nM was determined to be the most suitable experimental dosage for RNAi in subsequent examinations.
As a general rule, normal cell proliferation depends on orderly and efficient cell cycle process that ensures the duplication and transmission of genetic information from one cell generation to the next. In other words, deregulation of cell proliferation always lies in an abnormal cell cycle. Under CAC1-silencing conditions, flow cytometry in our study revealed elevated proportion of cells in the G1/G0 phase and a reduced rate of cells in the S phase. In essence, CAC1 knockdown induced G1 cell cycle arrest in the AGS cells, which was consistent with the results from the HeLa cell line . CAC1 was also capable of binding to CDK2 and stimulating its kinase activity at the G1/S phase transition without greatly changing the expressions of cyclinA, cyclinE, cyclinD1, CDK2, RB, PTEN, and so on . It is supposed that CAC1 depression attenuates the activity of CDK2 and interrupts the G1-S transition.
Apoptosis is one of the basic biological phenomena characterized by a series of transformations in cell morphology . Furthermore, apoptosis in concert with cell proliferation forms a crucial balancing mechanism that manages a large number of physiological procedures such as tissue homeostasis and normal development . Under pathological circumstances, aberrant apoptosis usually contributes much to the initiation and progression of cancer [10–12] and even influence the sensitivity of cancer cells to therapeutic interventions .
The unequivocal activity of CAC1 in cell cycle regulation raised the possibility that it is, more or less, involved in the process of apoptosis. In the current study, the proportion of early/late apoptotic cells increased with cisplatin treatment, but increased even more so when CAC1 expression was concurrently inhibited by RNAi. That is to say, apoptotic indices of the cisplatin plus CAC1-siRNA group obtained a significant increase in comparison with those of the former three groups with intact CAC1. Cumulative data imply that CAC1 may weaken the anti-cancer effect of cisplatin by counteracting cisplatin-induced apoptosis.
Regulation of apoptosis, however, relies on a network of anti- and proapoptotic molecules such as BCL2 family . BCL2 (B cell CLL/lymphoma 2) is a proto-oncogene located in the chromosomal region 18q21.3, which codes for an antiapoptotic 26-kDa protein containing four BH domains (BH1 ~ BH4)) . It can hinder the release of cytochrome c from the mitochondria, thus abrogating the activation of caspases and finally inhibiting apoptosis . According to early studies, the overexpression of BCL2 drives cells toward malignant transformation  and predicts the prognosis in many malignancies [18–22]. Particularly in gastric cancer, frequent expression of the BCL2 gene always occurred in malignant tissues [23–25]. High expression levels of the BCL2 gene, though correlated with less aggression of stomach cancer , had a lot to do with drug resistance of the cancer cells .
BAX (BCL2 associated X protein) gene is located in the human chromosomal region 19q13.3-q13.4 . Its 21-kDa encoding protein, in particular, serves as a proapoptotic member of the BCL2 family. BAX protein consists of three BH domains (BH1, BH2, and BH3) and shares a lot of homology with the BCL2 protein. Indeed, BAX protein acts as a suppressor of BCL2 to accelerate apoptotic cell death, by forming BAX/BCL2 complexes or by competing with other BCL2 targets . Overexpression of the BAX gene had a negative effect on cell growth in human gastric cancer, owing to the induction of apoptosis and to the enhancement of cell chemosensitivity . On the contrary, suppression of BAX gene expression induced tumorigenesis in gastric epithelia .
Cisplatin is a kind of chemotherapeutic agent widely used in solid malignancies including gastric cancer. It is generally accepted that its primary cytotoxic effect is DNA damage and subsequent induction of apoptosis , so variances of apoptosis-associated genes in cisplatin treated cells penetratingly mirror the mechanisms underlying cisplatin-induced apoptosis. As for our study, CAC1 expression was upregulated by cisplatin treatment, but was markedly downregulated by siRNA treatment despite previous cisplatin. Furthermore, underlying the increase of cisplatin-induced apoptosis that follows CAC1 silencing are concomitant gene expression alterations including the upregulation of P53 and BAX as well as the downregulation of BCL2. These effects suggest that CAC1 strengthens cisplatin-induced apoptosis by modulating the expression of BCL2, BAX and P53.
As mentioned previously, BCL2 is seemingly situated at the convergence of a couple of apoptotic pathways, and the ratio of BCL2 to BAX protein appears to be the final determinant of whether a cell enters the execution phase . In fact, it is the BCL2/BAX ratio that governs the sensitivity of cells to apoptotic stimuli [32, 33]. In the process of cisplatin-induced apoptosis, CAC1 might protect AGS cells from apoptosis by altering BCL2/BAX ratio, for CAC1 silencing brought out a pronounced increase of BAX and a decrease of BCL2, which was conducive to the occurrence of cell apoptosis.
Interestingly, the P53 gene in the AGS cells was upregulated with the cisplatin treatment, especially with synchronous suppression of CAC1. It is well known that P53 plays an important role in the management of cell cycle and apoptosis. DNA damage resulting from cisplatin may stimulate expression of the P53 protein that results in both expression of downstream P21 protein and G1 cell cycle arrest . If confronted with irreparable DNA damage, the P53 protein triggers programmed cell death . During cell apoptosis, P53 activates BAX via transcriptional  or transcription-independent  mechanisms, and represses transcription of BCL2. Furthermore, P53 can nontranscriptionally induce apoptosis . Therefore, CAC1 inhibition in the AGS cells can bring about excessive P53 accumulation, BAX accumulation, and BCL2 reduction, which ultimately potentiated apoptosis.
The mechanism by which CAC1 functions is not fully clarified. CAC1 was able to reinforce the activity of CDK2, and CAC1 silencing probably impaired the CDK2 activity. An early study argued that CDK2 inhibition could lead to ATM- and ATR-dependent P53 phosphorylation at serine 15, and thereby cause a significant increase of P53 and P21 protein . With the potential to activate CDK2, CAC1 is inclined to interfere with the P53 P21 pathway, and thus help AGS cells to resist G1 arrest and apoptosis. On the other hand, CAC1 can serve as a corepressor of RARα to negatively regulate retinoid acid-induced cellular differentiation and CoRNR box is confirmed to be a major functional region of CAC1. So it seems that CoRNR box has the potential to regulate cell proliferation and apoptosis, which deserves to be further investigated in other studies.