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. 2015 May 20;141(11):2037–2045. doi: 10.1007/s00432-015-1990-6

Synergistic anti-tumor efficacy of doxorubicin and flavopiridol in an in vivo hepatocellular carcinoma model

Min-Sun Kwak 1,2,#, Su Jong Yu 1,#, Jung-Hwan Yoon 1,✉, Sung-Hee Lee 1, Soo-Mi Lee 1, Jeong-Hoon Lee 1, Yoon Jun Kim 1, Hyo-Suk Lee 1, Chung Yong Kim 1
PMCID: PMC11823803  PMID: 25989942

Abstract

Purpose

A previous study showed that flavopiridol increased doxorubicin sensitivity in hypoxic hepatocellular carcinoma (HCC) cells by increasing apoptosis through suppressing hypoxia-inducible N-myc downstream-regulated gene-1 (NDRG1) expression. However, this has not been investigated in an in vivo HCC model. Therefore, we aimed to elucidate whether the combination of doxorubicin and flavopiridol has a synergistic anti-tumor effect in an in vivo HCC model.

Methods

An HCC mouse model was established by implanting C3H/He mouse with MH134 cells. Then, doxorubicin with or without flavopiridol was injected. The anti-tumor efficacy was assessed by evaluating tumor volumes, and the underlying mechanism was investigated by quantifying apoptotic cells, the Ki-67 proliferation index, and microvessel densities (MVDs). Immunohistochemistry of NDRG1 was performed to determine the underlying mechanism.

Results

Tumor growth was significantly suppressed in the doxorubicin + flavopiridol combination group compared to the other three groups. The percentage of apoptotic cells was significantly higher, and Ki-67-positive proliferating cells were significantly lower in the combination group compared to the other groups; however, MVDs were not significantly different across the groups. Increased apoptosis by flavopiridol occurred by suppressing hypoxia-inducible NDRG1 expression.

Conclusions

These results show that a combination of doxorubicin and flavopiridol has a synergistic anti-tumor effect in an in vivo HCC model. This synergistic effect of combination therapy was attributed to increased apoptosis and decreased proliferation of tumor cells rather than decreased angiogenesis. These findings suggest that flavopiridol might be an effective adjuvant therapy to doxorubicin-resistant HCC cells by inducing apoptosis through suppression of NDRG1 expression.

Keywords: Hepatocellular carcinoma, Doxorubicin, Flavopiridol, Apoptosis, Hypoxia

Introduction

Hepatocellular carcinoma (HCC), the fifth most common cancer worldwide, results in nearly one million deaths a year (Fattovich et al. 2004). HCC usually has hypervascular characteristics; thus, transarterial chemoembolization (TACE) is considered to be a standard treatment when radical therapies for HCC are not feasible. However, TACE is occasionally ineffective for advanced infiltrative HCCs or locally recurrent nodules after TACE. In these cases, systemic chemotherapy is considered as an alternative option. However, systemic chemotherapy is unfortunately mostly ineffective. Doxorubicin, an anthracycline agent that has been commonly tried for advanced HCC, showed a response rate of only approximately 15–20 % (Cao et al. 2012; Yeo et al. 2005). Therefore, there is a need to develop a more efficient strategy to improve treatment outcomes of doxorubicin treatment in HCC.

Efforts to increase doxorubicin sensitivity have been investigated using several approaches. Several factors have been suggested as possible underlying mechanisms of doxorubicin resistance in HCC (Fan et al. 2013; Xiang et al. 2014; Zhou et al. 2012). Among them, N-myc downstream-regulated gene-1 (NDRG1), a hypoxic inducible protein, is a factor that causes doxorubicin resistance in HCC, and the suppression of NDRG1 expression has been suggested as a strategy to increase doxorubicin sensitivity in HCC cells (Jung et al. 2010). Flavopiridol, a semisynthetic flavonoid, is an inhibitor of several cyclin-dependent kinases and has anticancer effects with various mechanisms of action, such as cyclin-dependent kinase inhibition, apoptosis induction, and DNA interaction (Zhai et al. 2002). In in vitro studies, flavopiridol enhanced doxorubicin sensitivity by increasing apoptosis of HCC cells through suppressing the expression of the hypoxia-inducible carcinogenesis-related NDRG1 protein, which is responsible for doxorubicin resistance in hypoxic HCC cells (Jung et al. 2010; Motwani et al. 2002). However, there has yet to be a study evaluating the synergistic effect of doxorubicin and flavopiridol in a mouse model of HCC.

Therefore, the purpose of this study was to evaluate the synergistic effect of doxorubicin and flavopiridol in an in vivo HCC mouse model and to determine the underlying mechanism.

Materials and methods

Cell culture and reagents

MH134 cells, a mouse HCC cell line, were used. MH134 cells were grown in RPMI-1640 medium supplemented with penicillin (100,000 U/L), streptomycin (100 mg/L), and 10 % fetal bovine serum. Overnight serum starvation was performed prior to all of the experiments to prevent the activation of serum-induced signaling. Doxorubicin and flavopiridol were purchased from Sigma-Aldrich.

Animals

Five-week-old male C3H/He mice were used for the animal experiments (Charles River Laboratories, Wilmington, MA, USA). The mice were kept under specific pathogen-free environment. All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Seoul National University Hospital (12-0176).

Mouse HCC model

An established subcutaneous mouse HCC model was used (Yamashita et al. 2001). Subcutaneous injections of 2.5 × 105 viable MH134 cells suspended in 0.1 mL of media were performed in the right flanks of mice, creating a bleb. When HCC tumor volumes reached 0.5–1.0 cm3, an intraperitoneal injection of doxorubicin (4 mg/kg) and/or flavopiridol (5 mg/kg) was performed daily for six consecutive days as follows. In the flavopiridol group, flavopiridol (5 mg/kg/d) was injected every other day for 3 days. In the doxorubicin group, doxorubicin (4 mg/kg) was injected every other day for 3 days. In the combination group, flavopiridol (5 mg/kg/day, on days 1, 3, and 5) and doxorubicin (4 mg/kg/day, on days 2, 4, and 6) were administered on alternating days. The control group was administered a 1.0 % DMSO/sodium chloride solution without drugs. The adequate dose of medication was based on a previous in vivo study (Budak-Alpdogan et al. 2009). The mice were monitored daily for tumor volume, body weight, behavior, and water/food consumption. Seven days after administering doxorubicin and/or flavopiridol, the mice were killed under isoflurane inhalation general anesthesia by exsanguination via cardiac puncture. HCC tumor masses were removed, formaldehyde-fixed, and cryopreserved.

Tumor growth kinetics

Tumor growth kinetics was described by an exponential model, and the following equation was used: V = V0 × exp (k × t) (Kim et al. 2009; Yu et al. 2012). The k is the growth rate constant associated with doubling time of HCC, V0 is the baseline tumor volume, and V is tumor volumes after t days. A software program of nonlinear mixed effect modeling (NONMEM) (version V, level 1.1, double precision) was used to analyze data, using the first-order conditional estimation method and the PRED routine (Beal 1984). Exponential random effect models were used to model inter-individual variabilities for k and V0. For example, the baseline tumor volume was modeled as V0i=V0×exp(ηi), where V0 indicates the typical value for baseline tumor volume for the population and V0i indicates the baseline tumor volume for an individual i. The ƞi is a random variable with normal distribution with mean 0 and variance ωV02. Additionally, the combination of additive and proportional error model represented as Yij=Y^ij×(1+εija)+εijp was used to model residual variability. In this model, Yij and Y^ij represent jth observed and predicted tumor volume in individual i, respectively, and εija and εijp are random variables of normal distribution with mean 0 and variances σa2 and σp2, respectively, for measurement j in individual i.

Apoptosis

TUNEL staining by ApopTag In Situ Apoptosis Detection Kits (Millipore) was performed to assess apoptosis in HCC tumor tissue. Six high-power fields (400×) with random selection were investigated, and positive TUNEL cells were counted and averaged. Apoptosis indexes refer to the percentage of apoptotic cells of the total number of cells.

Immunostaining of Ki-67

Immunohistochemical staining using Vectastain Elite ABC Kits (Vector Laboratories) was performed for mouse Ki-67 (BD Biosciences). Immunohistochemical scoring was performed without prior knowledge of the treatment regimen. Ki-67 positivity was quantified by counting a minimum of six randomly selected high-power fields (400×) and calculated as the percentage of positively stained cells to total cells using Aperio ImageScope (Aperio Technologies). This analysis was referred to as the proliferation index.

Microvessel density (MVD)

Immunohistochemical staining was performed for CD31 (Vector Laboratories) using Vectastain Elite ABC Kits (Vector Laboratories). Six randomly selected high-power fields (400×) were evaluated, and CD31-positive microvessels were counted and averaged in the most vascular regions of HCC tissues. Mean MVD of HCC was assessed as the number of microvessels/mm2.

Quantitative assessment of the central necrosis

Entire tissue sections of the tumor tissues were stained by hematoxylin and eosin (H&E), scanned at 40× magnification by ScanScope CS (Aperio Technologies) and then saved as digital images to measure the percentage of necrosis. Boundaries were drawn around the whole area of HCC and the region-dividing interface between viable tissue and necrotic areas using Aperio ImageScope (Aperio Technologies). On the H&E-stained slides, the pink amorphous regions with a glassy homogeneous appearance were considered to exhibit areas with necrosis, and purple pixel regions were regarded as viable tissues (Zhou et al. 2010). The percentage of central necrosis was defined as the necrotic area divided by the total tumor area (Zhou et al. 2010).

Immunohistochemical staining of NDRG1, hypoxia-inducible factor-1α (HIF-1α), and hexokinase II (HK II)

Immunohistochemical staining for mouse NDRG1 (Cambridge), HIF-1α (Santa Cruz), and goat HK II (Santa Cruz) was performed on HCC tumor tissue using the Vectastain Elite ABC Kit (Vector Laboratories). Positivity of NDRG1, HIF-1α, and HK II was quantified with Aperio ImageScope (Aperio Technologies) in six randomly selected high-power fields (400×) and averaged. Aperio ImageScope calculated the extent of the strongly positive, positive, weakly positive, and negative staining. Positivity of NDRG1, HIF-1α, and HK II was defined as the percentage of positively stained (strongly positive, positive, weakly positive) cells divided by the total cells.

Statistical analysis

The average results of three independent experiments are represented as the mean ± standard deviation (SD). An analysis of variance with post hoc analysis was performed to compare the quantitation of the necrotic area and the immunohistochemical staining (TUNEL assay, Ki-67, MVD, NDRG1, HIF-1α, and HK II) among the groups. To reduce the type I error, the LSD method was used in the post hoc analyses. All of the statistical analyses were performed with SPSS, version 19.0 (SPSS, Inc., Chicago, IL, USA) and Stata version 12.1 (StataCorp, College Station, TX, USA). P values <0.05 were considered statistically significant.

Results

The synergistic anti-tumor efficacy of doxorubicin and flavopiridol in the in vivo HCC mouse model

We evaluated the synergistic anti-tumor effect of doxorubicin and flavopiridol in the in vivo HCC mouse model. Indeed, the mean tumor volume was reduced significantly in the doxorubicin + flavopiridol combination group compared with the control, doxorubicin-only-, or flavopiridol-only-treated mice (Fig. 1). When exponential tumor growth model was assumed to evaluate the synergistic effect of doxorubicin and flavopiridol, the k (growth rate constants) were significantly lower for the doxorubicin + flavopiridol-treated group than for the untreated groups (p < 0.001), doxorubicin-only- (p = 0.007), or flavopiridol-only-treated mice (p = 0.001). This result demonstrates that doxorubicin and flavopiridol had a synergistic effect on the suppression of tumor growth (Table 1). Body weight change (p = 0.707), behavior, and water/food consumption were not different across the four groups.

Fig. 1.

Fig. 1

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Synergistic anti-tumor effect of doxorubicin and flavopiridol in the in vivo model of HCC. An in vivo HCC model is established in C3H/He mice by subcutaneous injection of MH134 cells. Doxorubicin and/or flavopiridol are injected into the mice for 6 days when the volumes of HCC had come to 0.5–1.0 cm3. The calculation of tumor volumes is according to the following formula: 0.5 × L (cm) × W2 (cm2), in which L indicated the maximum length and W indicated the maximal width. Seven to eight mice are included in each group by random allocation. The results are expressed as mean values ± SD

Table 1.

Tumor growth kinetics model generation by approaches of forward addition and backward elimination

Hypothesis −2 * log-likelihood df Diff (−2 * log-likelihood) Chi-square (α = 0.05) p value (adjusted p value)* Conclusion
Base model
K values of the four groups were the same. (k1 = k2 = k3 = k4) −88.0 2
Full model
Was k different according to drug group? −112.3 5 −24.3 7.81 (df = 3) <0.001 Yes
Backward elimination from the full model
Was k different between group 1 and group 2? −112.1 4 0.2 3.84 (df = 1) 0.655 (0.998) No
Was k different between group 1 and group 3? –107.1 4 5.2 3.84 (df = 1) 0.023 (0.128) No
Was k different between group 1 and group 4? −92.2 4 20.1 3.84 (df = 1) <0.001 (<0.001) Yes
Was k different between group 2 and group 3? −108.2 4 4.1 3.84 (df = 1) 0.043 (0.231) No
Was k different between group 2 and group 4? −93.0 4 19.3 3.84 (df = 1) <0.001 (0.001) Yes
Was k different between group 3 and group 4? −101.7 4 10.6 3.84 (df = 1) 0.001 (0.007) Yes
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Group 1 = control, group 2 = flavopiridol, group 3 = doxorubicin, group 4 = combination

* Adjusted p value by Sidak method

The synergistic anti-tumor effect of doxorubicin and flavopiridol is mediated by increased apoptosis and decreased tumor cell proliferation

To identify the mechanism underlying the synergistic effect of doxorubicin and flavopiridol, we quantitatively compared the level of apoptosis, tumor cell proliferation, and angiogenesis, which are well-known regulators of tumor progression, in the four groups. We evaluated the apoptotic levels in the four groups with TUNEL staining of the tumor tissues. The apoptotic indices were 1.5, 3.3, 3.2, and 5.1 % in the control, doxorubicin, flavopiridol, and combination groups, respectively. The percentages of TUNEL-positive cells were significantly high in the doxorubicin + flavopiridol combination group compared with the other three groups (p < 0.001, Fig. 2). When tumor cell proliferation was evaluated in an in vivo model of HCC by comparing Ki-67 proliferation indices, there was decreased proliferation in the combination group compared to the doxorubicin-only-treated group (p = 0.022, Fig. 3). However, the anti-angiogenic effect evaluated by MVD demonstrated no significant differences across the four groups (p = 0.478, Fig. 4).

Fig. 2.

Fig. 2

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Flavopiridol enhances doxorubicin-induced intra-tumoral apoptotic cell death. a Apoptosis in the HCC tissues is evaluated by TUNEL staining. TUNEL-positive cells are counted in six different randomly selected high-power fields (×400) and then averaged. b The apoptotic index, defined as TUNEL-positive cell percentages of the total cells, is used. The apoptotic index (proportions of TUNEL-positive cells) is significantly increased in the doxorubicin + flavopiridol combination group compared to the other three groups (p < 0.001). The results are expressed as mean values ± SD. *p < 0.001

Fig. 3.

Fig. 3

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Tumor cell proliferation is suppressed in the combination group compared to the doxorubicin-treated group. a Cell proliferation in HCC tumor tissues is determined by Ki-67 staining. b Ki-67 proliferation index is significantly lower in the combination group compared to the group treated with doxorubicin (p = 0.022). The results are expressed as mean values ± SD

Fig. 4.

Fig. 4

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No effect of doxorubicin or flavopiridol on intra-tumoral angiogenesis. Intra-tumoral microvessel densities (MVDs) are evaluated by immunohistochemical staining. The mean MVDs are evaluated by counting and calculating the average of CD31 + vessels in six randomly selected high-power fields (×400). No significant difference is noted among the four groups (p = 0.478). The results are expressed as mean values ± SD

The increased apoptosis by doxorubicin and flavopiridol might be through the suppression of NDRG1 expression

Hypoxia induces NDRG1 expression. Thus, we investigated whether an intra-tumoral hypoxic condition was achieved in the mouse model by evaluating necrotic areas and hypoxia-inducible expression of HIF-1α and HK II in tumor tissue. Central necrosis was observed in all of the HCC tissues from the mice, and the proportion of central necrosis was higher for the mice treated with the doxorubicin + flavopiridol combination compared with the control, doxorubicin, or flavopiridol groups (p = 0.004, p = 0.037, and p = 0.005, respectively, Fig. 5). HIF-1α (p = 0.270) and HK II (p = 0.481) were expressed similarly in all four groups, suggesting the existence of a hypoxic microenvironment in all of the tumor tissue (Fig. 6). Consistent with previous in vitro results (7), flavopiridol combined with doxorubicin significantly suppressed hypoxia-inducible NDRG1 expression in vivo HCC tissues compared to the control and doxorubicin-only-treated groups (p < 0.001 and p = 0.009, respectively, Fig. 7).

Fig. 5.

Fig. 5

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Central necrosis of mouse HCC tissue. a On the H&E-stained slides, central necrosis (region denoted as N) developed within the HCC tissue in the control, flavopiridol-only, doxorubicin-only, and doxorubicin + flavopiridol groups (×40). b Central necrosis is measured by necrotic region divided by the total HCC tumor area. The central necrosis area is significantly higher in mice treated with the doxorubicin + flavopiridol combination compared to the control, doxorubicin-only- and flavopiridol-only-treated mice (p < 0.05). N indicates necrotic area. V indicates viable area. The results are expressed as mean values ± SD. *p < 0.05

Fig. 6.

Fig. 6

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Expression of HIF-1α and HK II in mouse HCC tissue. Immunohistochemical staining for mouse HIF-1α and HK II is performed on paraffin-embedded mouse HCC tissues. Expression of HIF-1α and HK II is evaluated in six randomly selected high-power fields (×400), and the average is calculated. Positivity of HIF-1α and HK II is quantified using Aperio ImageScope (Aperio Technologies). a HIF-1α expression is similarly elevated across the four groups (p = 0.270). b HK II expression is similarly elevated across the four groups (p = 0.481). The results are expressed as mean values ± SD

Fig. 7.

Fig. 7

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Flavopiridol suppresses the expression of NDRG1 in mouse HCC tissue. a Immunohistochemical staining for mouse NDRG1 is performed on paraffin-embedded mouse HCC tissues. NDRG1 expression is evaluated in six high-power fields (×400) with random selection, and the average is calculated. NDRG1 positivity is quantified using the Aperio ImageScope (Aperio Technologies). The Aperio ImageScope calculated the extent of strongly positive, positive, weakly positive, and negative staining. NDRG1 positivity is defined as the percentage of positively stained (strongly positive, positive, weakly positive) cells divided by the total cells. b NDRG1 expression is significantly lower in the groups treated with flavopiridol compared to the control (p < 0.001) and groups treated with doxorubicin (p < 0.05). NDRG1 expression is also decreased in the combination group compared to the group treated with doxorubicin (p < 0.05). The results are expressed as mean values ± SD. *p < 0.05 and **p < 0.001

Discussion

The main finding of this study is the synergistic anti-tumor effect of doxorubicin and flavopiridol in an in vivo HCC model. Although a synergistic effect was previously investigated in an in vitro study, there has been no in vivo study on the effect of doxorubicin and flavopiridol on HCC. This is the first in vivo study demonstrating the synergistic anti-tumor efficacy of doxorubicin and flavopiridol in HCC. This synergistic effect was attributed to the enhanced apoptosis and decreased tumor cell proliferation. Flavopiridol induced tumor cell apoptosis by inhibiting hypoxia-inducible NDRG1 expression.

Doxorubicin, an anthracycline chemotherapeutic drug, inhibits DNA/RNA synthesis by intercalation between base pairs of DNA strands. The effect of doxorubicin was insufficient as a systemic chemotherapeutic agent in palliative treatment for advanced HCC (Asghar and Meyer 2012; Lai et al. 1988). In this in vivo study, tumor volume was decreased in the doxorubicin-only-treated group compared to the control group; however, this difference was not significant after adjusting for confounding variables (Table 1). The previous in vitro study showed that HCC cells in the hypoxic condition are more resistant to doxorubicin compared to normoxic HCC cells (Jung et al. 2010). Therefore, it can be hypothesized that doxorubicin-sensitive cells were killed by doxorubicin treatment, reducing the HCC tumor volume partially; however, doxorubicin-resistant cells under hypoxic conditions remain viable, making the effect of doxorubicin therapy insufficient. The addition of flavopiridol to doxorubicin enhanced cytotoxicity against those doxorubicin-resistant HCC cells and consequently further decreased tumor volume.

Flavopiridol is known to have several functions including cyclin-dependent kinase inhibition, apoptosis induction, and DNA interaction. In this study, flavopiridol single treatment suppressed tumor growth and caused apoptosis of HCC cells. When administered with other chemotherapeutic agents, flavopiridol can potentiate the efficacy of chemotherapeutic agents in several types of cancer cells (Li et al. 2001; Motwani et al. 2002; Wall et al. 2003; Zhai et al. 2002). In HCC cells, flavopiridol is known to enhance doxorubicin cytotoxicity (Jung et al. 2010), and another study also demonstrated flavopiridol sensitivity in HCC cells that were treated with and eventually became refractory to doxorubicin (Richard et al. 2005). In this HCC mouse model, the synergistic anti-tumor effect of doxorubicin and flavopiridol was attributed to the increased apoptosis.

Among diverse pathways associated with flavopiridol (Newcomb 2004), NDRG1 is one of these pathways. In colon cancer cells, NDRG1 was a target molecule modulating sensitivity to a chemotherapeutic agent, and flavopiridol suppressed NDRG1 (Motwani et al. 2002). Similarly, in vitro Huh-7 HCC cells, the suppression of NDRG1 expression by flavopiridol increased doxorubicin sensitivity in HCC cells via a caspase-dependent apoptosis pathway (Jung et al. 2010). NDRG1 is associated with aggressive HCC features in HCCs. High expression of NDRG1 was related to the metastasis, vascular invasion, and shorter overall survival in HCC (Chua et al. 2007). Furthermore, silencing NDRG1 reduced invasion and proliferation in vitro and inhibited in vivo tumor growth (Akiba et al. 2008; Melotte et al. 2010; Yan et al. 2008). An in vitro study demonstrated the association between increased NDRG1 expression and doxorubicin resistance and the association between suppressed NDRG1 expression with increased doxorubicin sensitivity; both associations indicate that NDRG1 is one of the molecules causing doxorubicin resistance. NDRG1 is induced under hypoxic conditions, which is the usual microenvironment inside solid tumors and is related to the tumor resistance to chemotherapy (Jung et al. 2010). An intra-tumoral hypoxic environment in this in vivo model induced NDRG1 expression, and flavopiridol significantly suppressed NDRG1 expression in the in vivo HCC tissue. This supports the hypothesis that suppressing NDRG1 by flavopiridol is a mechanism to overcome HCC resistance to doxorubicin under hypoxic conditions. Although a pathway downstream of NDRG1 was not evaluated in this study, a previous in vitro study demonstrated that glutaredoxin-2 is a downstream molecule of NDRG1-dependent resistance that contributes to HCC cell cytotoxicity (Jung et al. 2010). Glutaredoxin-2 is a known thiol/disulfide oxidoreductase component of the glutathione system and plays an important role in the regulation of mitochondrial reduction and oxidation system and cell death at the level of mitochondrial checkpoint (Gladyshev et al. 2001; Lillig et al. 2004). A previous study showed that the selective suppression of glutaredoxin-2 increased doxorubicin sensitivity (Lillig et al. 2004) and that glutaredoxin-2 expression was suppressed when NDRG1 siRNA or flavopiridol was administered to HCC cells (Jung et al. 2010). These results suggest that flavopiridol suppresses NDRG1 and the downstream glutaredoxin-2 molecule, which then increases the susceptibility of HCC cells to doxorubicin.

There are several limitations in this study. First, we used a subcutaneous HCC model, not orthotopic tumor model. As orthotopic tumor model would be more similar to the tumor microenvironment in clinical settings, further study with model of this type is warranted (Bagi et al. 2012). Second, to determine the exact role of NDRG1, overexpression or inhibition of NDRG1 expression is needed, but we did not show this result in this in vivo study. However, we already presented the role of NDRG1 in siRNA-mediated inhibition in a previous in vitro study (Jung et al. 2010).

In conclusion, this study shows that the combination treatment of doxorubicin and flavopiridol has a synergistic anti-tumor effect in an in vivo HCC mouse model. This effect is attributed to increased apoptosis and decreased proliferation of HCC tumor cells rather than suppressed angiogenesis. Increased sensitivity of HCC to doxorubicin was induced by suppressing hypoxia-inducible NDRG1 expression using flavopiridol. These results suggest that flavopiridol might be an effective adjuvant therapy to doxorubicin-resistant HCC cells by inducing apoptosis and inhibiting proliferation through the suppression of hypoxia-inducible NDRG1 expression.

Acknowledgments

This study was funded by the Liver Research Foundation of Korea. The funding organizations had no role in the study design; collection, analysis, and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. Authors have full control of all primary data and that authors agree to allow the journal to review these data if requested.

Conflict of interest

The authors declare that they have no conflict of interest.

Abbreviations

HCC

Hepatocellular carcinoma

TACE

Transarterial chemoembolization

NDRG1

N-myc downstream-regulated gene-1

NONMEM

Nonlinear mixed effect modeling

MVD

Microvessel density

H&E

Hematoxylin and eosin

HIF-1α

Hypoxia-inducible factor-1α

HK II

Hexokinase II

SD

Standard deviations

Footnotes

Min-Sun Kwak and Su Jong Yu have contributed equally to this article.

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