Farnesiferol c induces apoptosis via regulation of L11 and c-Myc with combinational potential with anticancer drugs in non-small-cell lung cancers

Though Farnesiferol c (FC) has been reported to have anti-angiogenic and antitumor activity, the underlying antitumor mechanism of FC still remains unclear. Thus, in the present study, we investigated the apoptotic mechanism of FC in human H1299 and H596 non-small lung cancer cells (NSCLCs). FC significantly showed cytotoxicity, increased sub-G1 accumulation, and attenuated the expression of Bcl-2, Bcl-xL, Survivin and procaspase 3 in H1299 and H596 cells. Furthermore, FC effectively suppressed the mRNA expression of G1 arrest related genes such as Cyclin D1, E2F1 transcription factor and CDC25A by RT-PCR. Interestingly, FC inhibited the expression of c-Myc, ribosomal protein L11 (L11) and nucleolin (NCL) in H1299 and H596 cells. Of note, silencing of L11 by siRNA transfection enhanced the expression of c-Myc through a negative feedback mechanism, while c-Myc knockdown downregulated L11 in H1299 cells. Additionally, combined treatment of FC and puromycin/doxorubicin promoted the activation of caspase 9/3, and attenuated the expression of c-Myc, Cyclin D1 and CDK4 in H1299 cells compared to single treatment. Taken together, our findings suggest that FC induces apoptosis and G1 arrest via regulation of ribosomal protein L11 and c-Myc and also enhances antitumor effect of puromycin or doxorubicin in NSCLCs.

Lung cancer is the leading cause of cancer related death all over the world and its main primary types are small lung cancer (10~15%) and non-small lung cancer (85~90%) 1,2 . In general, the treatment for lung cancer is surgery, chemotherapy, radiotherapy and targeted therapy mainly for EGFR or NF-κB 3 .
It was well documented that c-Myc is involved in proliferation, apoptosis, tumorigenesis, and cell cycle progression as one of the most frequently activated oncogene in human lung cancers [4][5][6][7] . Also, c-Myc was known to be regulated by ribosomal biogenesis related proteins, including L11, RPL5 and RPS14 [8][9][10] . Especially, L11 was known to act as a novel c-Myc inhibitor 8 . Recently many natural compounds are attractive due to their cancer chemopreventive effects and potential to synergize with classical anticancer agents [11][12][13] .
Farnesiferol C (FC), a compound isolated from Ferula assafoetida L. has been reported to have cytotoxic 14 and anti-angiogenic and antitumor effects 15 . Nonetheless, the underlying antitumor mechanism of FC was not fully understood so far. Thus, in the present study, the antitumor mechanism of FC was investigated in human H1299 and H596 non-small lung cancer cells (NSCLCs) in association with c-Myc and ribosomal protein L11 and also combinational potential of FC was examined with classical anticancer agents such as puromycin or doxorubicin in H1299 NSCLCs.

FC induces cytotoxicity and apoptosis in non-small lung cancer cells. Cytotoxicity of FC was eval-
uated in H1299 and H596 cells using MTT assay. As shown in Fig. 1B, FC significantly decreased the viability of H1299 and H596 cells. To examine whether or not the cytotoxic effect of FC is associated with apoptosis, cells were treated with various concentrations of FC in H596 and H1299 cells for 24 h. As shown in Fig. 1C, FC attenuated the expression of pro-caspase3, Bcl-2, Bcl-x L and Survivin in H596 and H1299 cells.
FC increases sub G1 population and attenuates the expression of Cyclin D1 and CDK4. To confirm the effect of FC on cell death and cell cycle arrest in cancer cells, H1299 cells were treated with various concentrations of FC for 24 h. The cells were stained with propidium iodide, and cell-cycle was analyzed by flow cytometry. As shown in Fig. 2A, FC increased sub G 1 population up to 5.39% and 16.14% by at the concentrations of 60 μM and 120 μM, respectively, compared to untreated control (2.83%) in H1299 cells. Consistently, FC treatment significantly decreased the expression of Cyclin D1 and CDK4 in H1299 and H596 cells, since Cyclin D1 binds and activates Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which regulate G1/S transition 16 (Fig. 2B). Also, mRNA levels of E2F1, CCND1, and transcription factor and cell division cycle 25 homolog A (CDC25A), which are related to G 1 phase 17 , were dramatically suppressed by FC treatment compared to untreated control (Fig. 2C).

Regulation of L11 and c-Myc is critically involved in apoptosis induced by FC in H1299
cells. Apoptosis is regulated by various proteins such as c-Myc, caspase3, and bcl-2 18,19 . FC dose-dependently suppressed the protein expression of c-Myc and its downstream NCL (nucleolin; c23) in H1299 cells, while it attenuated the expression of c-Myc alone in H596 cells (Fig. 3A). In contrast, FC attenuated the mRNA expression

FC promotes puromycin or doxorubicin-induced apoptosis in H1299 cells. Previous studies sug-
gested that puromycin enhanced apoptosis with melatonin in human leukemia HL-60 cells 20 . In the same line, to evaluate the combinational potential of FC with puromycin or doxorubicin, MTT assay and Western blotting were performed in H1299 cells. FC enhanced weak cytotoxicity of puromycin at a nontoxic concentration of 0.5 μg/ml in H1299 cells by combination treatment (Fig. 5A) Consistently, Western blotting showed that FC promoted weak apoptotic potential of puromycin to cleave caspase 9 and attenuate the expression of pro-caspase3, Bcl-2, Bcl-x L, Survivin Cyclin D1, CDK4 and c-Myc in H1299 cells ( Fig. 5B-D). In addition, we used 0.25 μM of doxorubicin for combination therapy with FC, since Rathos et al. reported the IC50 of doxorubicin is 0.23 μM in H1299 cells 21 . FC enhanced weak cytotoxicity and apoptotic activity of doxorubicin via the cleavage of caspase 3 and reduced expression of c-Myc in H1299 cells (Fig. 6A,B).

Discussion
Lung cancer still remains the leading cause of cancer-related mortality in the world. The majority of the lung cancer patients are the non-small cell (NSCLC) subtype 22 . Since, identification of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements in NSCLCs promoted targeted therapies 3,23 , several phytochemicals are attractive due to less toxicity and synergistic potential with classical anticancer agents 12,[24][25][26][27][28][29] .
Previously our group reported that FC had anti-angiogenic and antitumor activity targeting VEGFR1 or VEGFR2 signaling cascades. Nevertheless, the underlying antitumor mechanism of FC is not still unclear. Thus, in the current study, molecular antitumor mechanism of FC was examined in non-small lung cancer cells.
FC significantly exerted cytotoxicity, increased sub-G1 accumulation for apoptotic portion and attenuated the expression of survival genes such as Bcl-2, Bcl-x L , Survivin and pro-caspase 3 in H1299 and H596 cells, strongly implying the apoptotic activity of FC.
Cell cycle arrest is known as a stopping point in the cell cycle transition such as G0, G1, S and G2/M phases for cell duplication and division. Binding of Cyclin D1 and CDK4 or CDK6 was well known to trigger the transition from G1 to S phase 17,28,32 . Thus, cell cycle regulation is regarded as a good target for cancer therapy 30 . In the current study, FC effectively induced G1 arrest by suppressing G1/S transition phase related mRNA such as Cyclin The c-Myc is a multifunctional oncogene involved in cell growth, proliferation, tumorigenesis, and so is frequently upregulated in various types of cancer cells 31 . One of the key biological functions of c-Myc is to promote cell-cycle progression in several cancers 32 . After serum stimulation, c-Myc is induced at mRNA and protein levels and the cells enter the G1 phase of the cell cycle to promote biological processes including proliferation or apoptosis 33 . Interestingly, FC inhibited the expression of c-Myc and its downstream NCL at mRNA and protein levels in H1299 and H596 cells, demonstrating the role of c-Myc inhibition in FC-induced apoptosis and G1 arrest in NSCLCs. Also, it is noteworthy that FC reduced mRNA level of c-Myc by about 25%, while the protein level c-Myc was almost disappeared by FC, implying that FC works on c-Myc rather at posttranscriptional level, though further study is required in the near future.
Ribosomes are essential components of the protein synthesis machinery and ribosomal proteins play a critical role in cell proliferation, differentiation, apoptosis, DNA repair, and other cellular processes 10 . Among many ribosomal proteins, ribosomal protein L5 or L11 binds to c-Myc 8 and also activates TAp73 by overcoming MDM2 inhibition 9 , consequently leading to inhibition of c-Myc activity. Here, silencing of c-Myc promoted the ability of FC to exert cytotoxicity and sub G1 accumulation in H1299 cells, whereas L11 knockdown abrogated the antitumor activity of FC in H1299 cells. Of note, though FC downregulated the expression of not only c-Myc but also L11 compared to untreated control in H1299 cells, c-Myc knockdown enhanced the downregulation of L11 and silencing of L11 induced upregulation of c-Myc in H1299 cells, indicating the possible feedback regulation of c-Myc by L11. These results were supported by previous report 34 by Dai et al. that reduction of L11 by siRNA increases c-Myc levels through a negative feedback mechanism, since L11 directly binds to the Myc box II (MB II) for c-Myc-enhanced ribosomal biogenesis.
Recently combination therapy of anticancer agents and natural compounds are on the spotlight for reducing side effect and enhancing antitumor activity 35,36 . Here, combined treatment of FC and puromycin/doxorubicin promoted the activation of caspase 9/3, and attenuated the expression of c-Myc, Cyclin D1 and CDK4 in H1299 cells compared to single treatment, demonstrating the potential of using FC for combination therapy with classical anticancer agents such as puromycin or doxorubicin.
In summary, FC significantly showed cytotoxicity, increased sub-G1 accumulation, attenuated the expression of Bcl-2, Bcl-x L , Survivin and procaspase 3, suppressed the mRNA expression of G1 arrest related genes such as Cyclin D1, E2F1 and CDC25A, reduced the expression of c-Myc and NCL at mRNA and protein levels in H1299
RT-qPCR was operated with the light cycler TM instrument (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer's protocol. The mRNA level of GAPDH was used to normalize the expression of genes of interest.
RNA transfection assay. Cells were transiently transfected with a validated scrambled control small interfering (si)RNA, or siRNA specifically for L11 or c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA) or Flagtagged L11 overexpression vector kindly provided by Prof. Hua Lu (School of Medicine, Oregon Health and Science University, USA) by using Interferin TM transfection reagent (Polyplus-transfection Inc., New York, NY). Briefly, the mixture of siRNA or Flag-tagged L11 overexpression vector with Interferin TM transfection reagent was incubated for 10 min, added to each well of the cells (siRNA final concentration = 40 nM) and incubated at 37 °C for 48 h before drug treatment.
Statistical analyses. Data were presented as means ± standard deviation (SD) of three or more replicates.
Statistical significance was verified by Student's t-test using Sigma plot software (System Software Inc., San Jose, CA, USA).