Augmentation of differentiation and gap junction function by kaempferol in partially differentiated colon cancer cells

Yasushi Nakamura3, Chia-Cheng Chang1, Toshio Mori2, Kenji Sato, Kozo Ohtsuki, Brad L. Upham1 and James E. Trosko1

Department of Food Sciences and Nutritional Health, Kyoto Prefectural University, Shimogamo-Hangi, Sakyo, Kyoto 606-8522, Japan, 1 Department of Pediatrics and Human Development and National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824 1302, USA and 2 Radioisotope Research Center, Nara Medical University, 840 Shijo, Kashihara 634-8521, Japan

3 To whom correspondence should be addressed Email: yas{at}kpu.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Kaempferol induces differentiation in partially differentiated colon cancer cells which express low levels of connexin43 protein and connexin43 mRNA (KNC cells). Differentiation was observed as changes in cell morphology and the activity of alkaline phosphatase. Increased differentiation in kaempferol-treated KNC cells correlated with restoration of gap junctional intercellular communication (GJIC), increased levels of connexin43 protein and its phosphorylation status. Phosphorylation (activation) of Stat3 and Erk was also reduced by kaempferol. An inhibitor of Stat3 phosphorylation also induced morphological changes in KNC cells similar to those in kaempferol-treated cells, suggesting that kaempferol-induced differentiation may be mediated by inhibition of Stat3 phosphorylation. These effects were not observed in HCT116 cells, a poorly differentiated colon cancer cell line deficient in expression of connexin43 mRNA and connexin43 protein. In conclusion, kaempferol might function as an anticancer agent by re-establishing GJIC through enhancement of the expression and phosphorylation of connexin43 protein in a tumorigenic colon cancer cell line that already expresses connexin43 mRNA via a Stat3-dependent mechanism. In contrast, kaempferol had no effect in a tumorigenic colon cancer cell line that did not express connexin43 mRNA and was deficient in GJIC.

Abbreviations: ALPase, alkaline phosphatase; Cx43, connexin43 protein; cx43, connexin43 mRNA (GJA1); Erk, extracellular receptor kinase; FBS, fetal bovine serum; GJIC, gap junctional intercellular communication; PBS, phosphate-buffered saline; SFM, serum free medium; Stat3, signal transducer and activator of transcription 3


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carcinogenesis has been conceptualized as a multi-step, multi-mechanism process consisting of initiation, promotion and progression phases. While the exact mechanisms underlying each of these phases are not yet known, the reversible inhibition of gap junctional intercellular communication (GJIC) and apoptosis have been hypothesized to be involved in the tumor promotion phase (13). GJIC has been implicated in the ability of a cell to regulate growth control via adaptive responses: differentiation, proliferation and apoptosis (4). Accordingly, one strategy for efficacious chemoprevention and chemotherapy would be to prevent down-regulation of GJIC by tumor promoting chemicals and to restore GJIC in GJIC-deficient tumor cells (1,2). Gap junctions are channels between contiguous cells allowing the passive transfer of low molecular weight molecules (<1200 Da) and are made up of protein subunits termed connexins (5,6). Connexin genes have been shown to function as tumor suppressor genes (2,7). Transfection of connexin genes into neoplastic cells results in the restoration of GJIC and reversal of the transformed phenotype (813). Different species of connexin are selectively expressed in specific organs and cells, e.g. connexin43 protein (Cx43) predominantly plays a role in GJIC in rat liver epithelial cells.

Similarly, some natural anticarcinogenic compounds, such as carotenoids (14), caffeic acid phenethyl ester (15), flavonoids (apigenin and tangeretin) (16,17) and diallyl disulfide (18), are also known to prevent inhibition of GJIC by tumor promoters or to restore GJIC in neoplastic cell lines with expressed but non-functional connexins, resulting in reversal of the transformed phenotype. Kaempferol is a flavonoid widely distributed in the plant kingdom and which is found in high concentrations in cruciferous and liliaceous vegetables such as broccoli and onion. The consumption of flavonoids has been linked to the prevention and therapy of some human cancers.

Colon cancer is one of the most widely distributed cancers in the world. Because most cases have few to no symptoms, such as pain, the tumor is often only diagnosed in the later stages of the disease. Therefore, an appropriate therapeutic strategy rather than prevention is required in colon cancer. Conventional cancer therapy uses anticancer drugs to kill tumorigenic cells. Unfortunately, many patients suffer from the side-effects of these cytotoxic drugs. Therefore, alternative chemotherapies, such as the induction of differentiation and apoptosis of cancer cells are believed to be better approaches.

In this study we applied kaempferol to two human colon cancer cell lines having different profiles of expressed gap junction genes and function. One is a partially differentiated colon cancer cell type, KNC, which is deficient in GJIC and Cx43 expression but expresses connexin43 mRNA (cx43), while the other is a poorly differentiated colon cancer cell type, HCT116, which is deficient in GJIC and in Cx43 and cx43 expression. First, we showed that kaempferol induces a differentiation effect that is correlated with a cytostatic effect in KNC but not HCT116 cells. Second, kaempferol was found to concomitantly change the phosphorylation status of Cx43 to normal levels in KNC cells and also to decrease the phosphorylation of Stat3 and Erk. Further, inhibition of Stat3 phosphorylation by a selective inhibitor mimicked the kaempferol-induced differentiation effect in the tumorigenic cell line containing cx43 but not in the colon cancer cell line lacking cx43. Stat3 is a key signaling molecule for many cytokines and growth factor receptors and is involved in differentiation and apoptosis (1921). Stat3 is constitutively activated in many human epithelial malignancies (22). Regulation of the Jak/Stat3 signaling pathway by kaempferol might be one therapeutic strategy for colon cancer cells with expressed Cx43 and constitutively activated Stat3.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Kaempferol and Lucifer yellow CH were obtained from Sigma (St Louis, MO). AG490 (N-benzyl-3,4-dihydroxy-benzylidenecyanoacetamide) was purchased from Calbiochem (San Diego, CA) and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynylthio)butadiene] was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). A modified Eagle's medium (Formula no. 78-5470EF), the defined keratinocyte SFM and fetal bovine serum (FBS) were purchased from GIBCO-Invitrogen (Grand Island, NY). Anti-connexin43 rabbit polyclonal antibody (catalog no. 71-0700) was purchased from Zymed (South San Francisco, CA). Anti-Erk rabbit polyclonal antibody (catalog no. 9101), anti-phospho-Erk rabbit polyclonal antibody (catalog no. 9102), anti-Stat3 rabbit polyclonal antibody (catalog no. 9132), anti-phospho-Stat3 rabbit polyclonal antibody (catalog no. 9131) and anti-rabbit IgG horseradish peroxidase-linked secondary antibody (catalog no. 7074) were purchased from Cell Signaling Technology Inc. (Beverly, MA). The ECL western blotting detection kit and X-ray film were purchased from Amersham Life Science (Denver, CO).

Cells
The MSU-2 human foreskin fibroblast line was developed in our laboratory at Michigan State University. HCT116 (GJIC, Cx43-deficient, cx43-deficient) is a poorly differentiated human colon cancer cell line that was obtained from Dr Michael Brattain (Baylor College of Medicine). KNC (GJIC, Cx43-deficient, cx43-expressing) is a partially differentiated human colon cancer cell line obtained from surgically resected colon carcinoma tissue that was diagnosed as a well-differentiated colon carcinoma. KNC cells are anchorage independent, fibroblast growth factor dependent and are not immortalized. Modified Eagle's medium containing 10% fetal bovine serum was used to grow the MSU-2, HCT116, WB (rat liver epithelials), WBsrc (v-src oncogene-transfected WB cells), Ming and Capan-1 (human pancreatic cancer cells) cell lines. KNC cells were grown in defined keratinocyte SFM containing 10% fetal bovine serum.

Treatment of cells with kaempferol, U0126 and AG490
Cells (2 x 104), plated in 35 mm culture plates with 2 ml of medium overnight, were treated with kaempferol (in 10 µl of acetonitrile solvent), AG490 or U0126 (in 1 µl of DMSO solvent). The medium and chemical treatments were renewed every other day.

Cytotoxicity assay
Cells (4 x 104) were plated into 60 mm culture plates (3 plates per treatment). Twenty-four hours after plating the cells were incubated with acetonitrile (0.5%) or kaempferol (2.5, 5, 10 and 20 µM) for 24 h. After trypsinization with 0.25% trypsin and 1 mM EDTA, cells from each dish were counted using a hemacytometer.

Reverse transcription–PCR
RNA was isolated using an RNeasy kit (Qiagen), according to the manufacturer's instructions. RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). The cDNA obtained was then amplified for 24 cycles (15 s at 92°C, 30 s at 60.2°C and 1 min at 68°C) of PCR in the presence of Taq polymerase (Roche) in a total volume of 50 µl. The cx43 primer sequences used were 5'-TGGATTCAGCTTGAGTGCTG-3' (sense) and 5'-TCTTTCCCTTAACCCGATCC-3' (antisense). The PCR products were then resolved in a 2.0% agarose gel and the bands were treated with ethidium bromide and observed under 265 nm UV irradiation. The control GAPDH primers were 5'-CGACCACTTTGTCAAGCTCA-3' (sense) and 5'-AGGGGAGATTCAGTGTGGTG-3' (antisense).

Alkaline phosphatase (ALPase) activity
Cells were washed with phosphate-buffered saline (PBS) twice and lysed in a buffer containing 0.1 M carbonate and 2 mM MgCl2, pH 9.5. The lysate was sonicated and the protein was quantified using a DC assay kit (Bio-Rad, Richmond, CA). The lysate (20 µg protein) and 0.3 ml of ALPase substrate solution (0.3 mg p-nitrophenyl phosphate in 0.2 M Tris buffer; Sigma, St Louis, MO) were well mixed in a 1.5 ml plastic tube. The tube was incubated at 37°C for 15 min and 0.5 ml of 0.2 M NaOH was added to terminate the substrate–enzyme reaction. The ALPase activity was calculated by reading sample absorption at 405 nm from a linear standard equation derived from the absorption readings of consecutive known ALPase activities.

Cell–cell communication by scrape loading dye transfer assay
GJIC was measured using the scrape loading dye transfer technique (23). Briefly, following exposure to kaempferol the cells were washed three times with PBS. The fluorescent dye Lucifer yellow (Sigma), dissolved in PBS (1 mg/ml), was added to the cells. Multiple scrape lines were made by gently pushing a surgical blade across the cell monolayer to allow passage of the membrane impermeable dye into ruptured cells. After 3 min incubation the cells were washed with PBS to remove extracellular dye and were fixed with 4% formalin. Dye migration was observed and digitally photographed at 200x using a Nikon epifluorescence microscope illuminated with an Osram HBO 200 W lamp and equipped with a COHU video camera. The program Gel-Expert (Nucleotech, San Mateo, CA) was used to quantify GJIC by determining the distance of dye migration. The distance of dye migration perpendicular to the scrape line (i.e. between adjacent cells linked only by gap junctions) represents the ability of cells to communicate via GJIC. GJIC activity was calculated as a fraction of the solvent control. All treatments were tested with two perpendicular measurements in triplicate plates.

Western blotting
Proteins were extracted with 20% SDS solution according to the method described (23). The protein concentrations of the samples were determined with a DC assay kit (Bio-Rad, Richmond, CA). The proteins (7.5 µg) were separated on 7.5% (for Erk and Stat3) or 12.5% (for Cx43) SDS–PAGE (24), and transferred from the gel to PVDF membranes (Millipore Corp, Bedford, MA) (25). Cx43, Erk, phospho-Erk, Stat3 and phospho-Stat3 were detected with anti-connexin43 rabbit polyclonal antibody (Zymed, San Francisco, CA), anti-Erk, anti-phospho-Erk, anti-Stat3 and anti-phospho-Stat3 rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA), respectively, using anti-rabbit IgG horseradish peroxidase-linked secondary antibody (Cell Signaling Technology) and then observed using super signal west dura extended duration substrate (Pierce, Rockford, IL) and an ECL detection kit (Amersham Life Science, Denver, CO).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test of kaempferol cytotoxicity
The cytostatic and cytotoxic effects of kaempferol on MSU-2 human fibroblasts were determined by cell growth. MSU-2 cells showed an exponential rate of cell proliferation for 7 days when grown in the vehicle control (Figure 1). Kaempferol (2.5, 5, 10 and 20 µM) had no significant effect on the growth rate of MSU-2 cells (Figure 1) or on cell viability, whereas exposure to 40 µM kaempferol stopped cell growth (data not shown). As a consequence of these results we used kaempferol at doses between 2.5 and 20 µM in the following experiments.



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Fig. 1. Effect of kaempferol on the proliferation of fibroblasts and colon cancer cells. Samples of 1.0 x 104 cells were each seeded into 60 mm dish. Cell numbers were counted at 0, 1, 3, 5 and 7 days after addition of kaempferol ({circ}, 0 µM; •, 2.5 µM; {square}, 5 µM; {blacksquare}, 10 µM; {triangleup}, 20 µM). (A) MSU-2 human fibroblasts; (B) KNC partially differentiated colon carcinoma cells; (C) HCT116 poorly differentiated colon carcinoma cells. Each point represents the mean ± SD of triplicate experiments. *Significantly different from the control without kaempferol at P < 0.01 (paired t-test).

 
In KNC and HCT116 colon cancer cells kaempferol did not show any effect on cell viability (up to 10 µM), as determined by the lack of cell detachment from the culture plates. After kaempferol treatment the growth rate of KNC cells started to decline after 3 days, and the growth rate was greatly different from the control after 5 and 7 days treatment. In contrast, HCT116 cells responded to kaempferol by an alteration in the growth rate on day 1, but seemed to rapidly adapt thereafter, as can be seen from the parallel growth rate lines for 1–7 days treatment with or without kaempferol (20 µM). Kaempferol is consequently considered to be a cytostatic chemical for KNC but not for either MSU-2 or HCT116 cells.

Kaempferol-induced morphological changes in KNC cells
When KNC cells were cultivated for 10 days with kaempferol (20 µM), dramatic morphological changes were observed, including the appearance of neuron-like (Figure 2B) and triangular (Figure 2C) cells. When the dose of kaempferol was lowered to 2.5–10 µM, no morphological changes were seen in KNC cells (data not shown). HCT116 cells did not show any morphological changes at any dose (data not shown). Thus kaempferol (20 µM) induces dramatic morphological changes only in partially differentiated colon cancer cells (KNC) and not in the poorly differentiated colon cancer cells (HCT116).



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Fig. 2. Morphological changes induced by kaempferol in KNC cells after 10 days' treatment. KNC cells (3.0 x 103) were treated for 10 days with kaempferol (20 µM) in a 60 mm dish. (A) Normal morphology of KNC cells without kaempferol. Neuron-like (B) and triangular (C) cells were observed with kaempferol.

 
Kaempferol increases ALPase activity in KNC cells
An increase in ALPase activity preceded the differentiation of colon cancer cells. ALPase has been used as a marker in the early stages of differentiation of colon cancer cells (26). The partially differentiated colon cancer cell line KNC showed higher ALPase activity (17.15 ± 0.84 µU) than the poorly differentiated colon cancer cell line HCT116 (5.94 ± 0.33 µU) (Figure 3). When KNC cells were cultivated for 5 and 7 days in the presence of kaempferol the ALPase activity increased significantly. However, kaempferol had no effect on ALPase activity in HCT116 cells. These effects may be related to the ability of kaempferol to induce differentiation in partially differentiated colon cancer cells (KNC) but not in poorly differentiated colon cancer cells (HCT116).



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Fig. 3. Stimulation of ALPase activity by kaempferol in KNC but not HCT116 cells. ALPase activity was measured 1, 3, 5 and 7 days after addition of kaempferol (0, 0.5, 1, 3, 5, 10 and 20 µM). Each point represents the average ± SD (vertical error bar) of three experiments. {circ}, HCT116 poorly differentiated colon carcinoma cells; •, KNC partially differentiated colon carcinoma cells.

 
Kaempferol increases GJIC in KNC cells
GJIC has been hypothesized to play a role in the suppression of growth of initiated cells by neighboring cells in the tumor promotion step of carcinogenesis (2). In addition, GJIC is also considered to be an indicator for the degree of differentiation or malignancy of cancer cells. The relative level of GJIC in WB cells (a non-tumorigenic rat liver epithelial cell line serving as a positive control) was 1.38 times higher than KNC cells (Figure 4). In contrast, the level in HCT116 cells was 0.44 times less. These amounts correlated with the level of malignancy. Kaempferol was found to enhance the level of GJIC in KNC cells to 1.33 times (5 µM) and 1.29 times (10 µM) higher than the untreated cells. On the other hand, no enhancement of GJIC was detected in HCT116 cells following kaempferol treatment.



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Fig. 4. Restoration of GJIC by kaempferol in KNC cells. GJIC was measured 7 days after addition of kaempferol (0, 5 and 10 µM). Each point represents the average ± SD (vertical error bar) of three experiments. {circ}, HCT116 poorly differentiated colon carcinoma cells; •, KNC partially differentiated colon carcinoma cells; {blacksquare}, WB rat liver epithelial cells, as a positive control. (A) 0 µM kaempferol in WB cells; (B) 0 µM kaempferol in HCT116 cells; (C) 0 µM kaempferol in KNC cells; (D) 5 µM kaempferol in KNC cells; (E) 10 µM kaempferol in KNC cells.

 
Kaempferol increases Cx43 in KNC cells
The subunits of gap junctions, the hemi-channels or connexons, consist of six subunits of Cx43. Up-regulation of Cx43 expression can induce a significant enhancement of GJIC in KNC cells. Non-tumorigenic rat liver epithelial cells (WB cells) produced high levels of non-phosphorylated Cx43 (P0) and its phosphorylated forms (P1 and P2) (Figure 5). KNC cells express low levels of P0 Cx43 and very low levels of P1 and P2. Kaempferol in the dose range 5–20 µM increased total Cx43 and converted P0 Cx43 to the phosphorylated forms, which are important for functional GJIC in KNC cells. In HCT116 cells Cx43 level did not increase in response to kaempferol treatment. Thus, kaempferol increased the amount of Cx43 and its phosphorylated forms in Cx43-expressing but GJIC-non-functional KNC cells, but did not enhance GJIC in GJIC-deficient HCT116 cells.



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Fig. 5. Augmented levels of cx43 mRNA, Cx43 protein and phosphorylation of Cx43 in KNC cells treated with kaempferol. Cx43 protein and cx43 mRNA levels were measured 7 days after addition of kaempferol (0–20 µM). (A) Cx43; (B) cx43. P0, non-phosphorylated Cx43; P1 and P2, phosphorylated forms of Cx43.

 
Since kaempferol did not change the steady-state level of cx43 mRNA in KNC cells, functional GJIC is dependent on the ability of kaempferol to phosphorylate Cx43 and not to alter mRNA expression. In contrast, the lack of Cx43 in HCT116 cells is due to a lack of transcriptional expression of cx43 mRNA, leading to no functional GJIC (Figure 4).

Stat3 protein is highly phosphorylated in KNC cells
Stat3 is activated by tyrosine phosphorylation at Tyr705, resulting in homodimerization and migration into the nucleus (27). Tyr705-phosphorylated Stat3 protein was detected by western blotting (7.5% polyacrylamide gel) as a 90 kDa protein (Figure 6A). A comparative study of KNC cells and other tumorigenic cell lines (WBsrc, HCT116, Ming and Capan-1) showed that KNC cells express phosphorylated Stat3 protein at high levels (Figure 6A).



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Fig. 6. Kaempferol inhibits phosphorylation of Stat3 in KNC cells, which have high levels of expressed Stat3. (A) Stat3 protein was measured in WBsrc, HCT116, KNC, Ming and Capan-1 cells. (B) KNC cells were treated with kaempferol (0–20 µM) for 7 days. The densitometric values are from X-ray film exposed within the linear range to chemiluminescent developed western blots loaded with 7.5 µg protein. Phosphorylated Stat3 data were normalized to total Stat3.

 
Kaempferol inhibits phosphorylation of Stat3 in KNC cells
KNC cells were treated with 2.5–20 µM kaempferol for 7 days. Kaempferol did not change the constitutive level of Stat3 up to 10 µM, but it was reduced at 20 µM (Figure 6B). Tyr705-phosphorylated Stat3 was markedly reduced by treatment with 20 µM kaempferol. The relative band density of the Tyr705-phosphorylatd Stat3, normalized to that of the constitutively expressed Stat3 band, showed a dose-dependent decrease.

Kaempferol inhibits phosphorylation of Erk in KNC cells
KNC cells were treated with 2.5–20 µM kaempferol for 7 days. Kaempferol did not change the level of Erk1 and Erk2 up to 5 µM, but reduced them at 10 µM (Figure 7). Erk2 was highly phosphorylated compared with Erk1 in KNC cells. The phosphorylation of Erk1 and Erk2 was markedly reduced by 20 µM kaempferol. The relative band densities of phosphorylated Erk1 and Erk2, normalized to that of the constitutively expressed Stat3 band, showed dose-dependent decreases.



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Fig. 7. Kaempferol-induced phosphorylation of Erk in KNC cells. KNC cells were treated with kaempferol (0–20 µM) for 7 days. Cell lysate (7.5 µg protein) was used for western blot determination of Erk and its subsequent densitometric analysis. Phosphorylated Erk was normalized to total Erk.

 
A Stat3 phosphorylation inhibitor, AG490, changes the morphology of KNC cells
When KNC cells were treated with a Stat3 phosphorylation inhibitor, AG490 (25 µM), for 3 days, a morphological change (i.e. the appearance of triangular cells) was observed (Figure 8B). However, the Erk phosphorylation inhibitor U0126 (1 µM) did not induce any morphological change (Figure 8C). The triangular cells were observed with U0126 + AG490 (Figure 8D), indicating that U0126 did not interfere with the effect of AG490. This morphological change was not observed in HCT116 cells following U0126 and/or AG490 treatment (data not shown).



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Fig. 8. Effect of Erk and Stat3 phosphorylation inhibitors on morphological changes and GJIC. Cells (2.0 x 104) were seeded into 35 mm dishes. These were then treated with U0126 and/or AG490 for 3 days. Arrows show triangular cells. (A) Control; (B) 25 µM AG490; (C) 1 µM U0126; (D) 25 µM AG490 + 1 µM U0126.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study describes the effects of the plant flavonoid kaempferol on two human colon cancer cell lines, KNC cells (a partially differentiated cancer cell line that is deficient in GJIC and Cx43 expression but expresses cx43) and HCT116 cells (a poorly differentiated cancer cell line that is deficient in GJIC and the expression of Cx43 and cx43). The most significant finding of these studies is the ability of kaempferol to restore the normal phosphorylation status of Cx43 and Stat3 concomitant with the induction of cell differentiation and a cytostatic effect in KNC cells.

The cytostatic effect of kaempferol on the partially differentiated human KNC colon cancer cells (Figure 1) was cell type specific, since the same effective dose of kaempferol did not show any significant cytostatic and cytotoxic effects in human fibroblast cells. In contrast to KNC cells, the poorly differentiated human colon cancer cell line HCT116 did not show this kaempferol effect. Kaempferol was also capable of inducing morphological differentiation of KNC cells (Figure 2). A similar differentiation effect can be induced in KNC cells by treatment with the Stat3 phosphorylation inhibitor AG490 (Figure 8B and D). Since treatment of KNC cells with kaempferol decreased Stat3 phosphorylation, which is typically highly expressed in KNC as compared with HCT116 cells (Figure 6A and B), inhibition of Stat3 phosphorylation would appear to be involved in induction of the morphological changes seen in KNC cells. Although kaempferol reduced the levels of phosphorylated Erk, the lack of morphological changes during this diminished activity of Erk, as well as the lack of an effect on morphological differentiation by the Mek inhibitor U0126, indicate that the Erk pathway does not play a role in kaempferol-induced morphological differentiation of KNC cells.

GJIC has been implicated in cell growth control via adaptive responses: differentiation, proliferation and apoptosis (4). In this regard, our results show that kaempferol restores GJIC, induces a cytostatic effect and morphological differentiation and increases ALPase activity in KNC cells. The steady-state level of cx43 was not altered by kaempferol. Since Cx43 has a very short half-life (28), kaempferol might have the capability to increase translation of Cx43, as well as post-translational effects (i.e. protect against degradation by regulation of phosphatases and proteases that can digest Cx43) (29).

On the other hand, HCT116 cells, which do not transcriptionally express cx43, were not induced to differentiate by kaempferol. To suppress the growth of these Cx43-deficient HCT116 cancer cells, as well as other cancer cells that similarly do not transcriptionally express connexins (e.g. HeLa cells), the cells would first need to be treated with agents that could induce transcription of the connexin genes (30), followed by treatment with reagents, such as kaempferol, to induce expression of functional gap junction proteins.

In summary, our results show that kaempferol can induce ALPase activity, decrease Stat3 activity and restore Cx43 phosphorylation and functional GJIC, resulting in a cytostatic or differentiation effect in a human cancer cell line with expressed but non-functional GJIC (KNC cells). In contrast, kaempferol had no such effects on a human colon cancer cell line with no expressed Cx43 and functional GJIC. These results suggest that suppression of the Jak/Stat3 signaling pathway plays an important role in the differentiation of colon cancer cells with expressed cx43 but non-functional GJIC in response to kaempferol. Further, regulation of Jak/Stat3 signaling by other chemicals might provide a novel therapeutic strategy for some types of colon cancer, particularly for cancer cell types having constitutively activated Stat3. One of the major implications of these results is that no potential cancer chemopreventive or chemotherapeutic agent will work on all types of cancer cells, not even those within the same organ. In future studies the observed kaempferol effects should be confirmed in other colon cancer cell lines or different types of normal colon cells with different profiles of gap junction expression and differentiation.


    Acknowledgments
 
This research was partly supported by a NIEHS Superfund Basic Science Program grant to J.E.T. (PA42 ES04911).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 26, 2004; revised November 24, 2004; accepted December 7, 2004.