Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Abstract

Ciprofloxacin or enrofloxacin induces S-G₂/M arrest

Based on the similarity in sensitivity between canine OSA cell lines, we chose to focus mechanistic studies on Abrams cells. The Abrams cell line seemed ideal based on previously published positive characteristics of this cell line including in vivo growth and the ability to form pulmonary metastases in nude mice [14]. In order to explore the mechanism of FQ induced inhibition of OSA cells, we performed cell cycle analyses. CPFX and ENFX treatment of Abrams cells resulted in S-G₂/M phase cell cycle accumulation after exposure for 4 and 5 days at concentrations at or above 20 ug/ml (Table 1). In addition, CPFX or ENFX both increased the number of canine osteosarcoma cells in sub G₁ (Figure 2).

Figure 2: CPFX and ENFX -induced S-G₂/M arrest on in canine OSA cells.

The effect of CPFX (A) or ENFX (B) on cell cycle distribution was investigated by flow cytometry in Abrams cells following 4 days or 5 days of treatment. Decreases in the proportions of cells in the G₁ phase of the cell cycle and increases in the S and G₂ phases were observed. The actual percentage of gated cells are presented in Table 1.

Table 1: Percentage of gated cells in cell cycle analysis. CPFX and ENFX treated with 20 or 40 ug/ml for 4 or 5 days on Abram cell.

Altered expression of cdc2 and phosphorylated cdc25C in ciprofloxacin or enrofloxacin treated canine Abrams OSA cells

To further examine the mechanism behind S-G₂/M phase cell cycle accumulation we examined the effects of FQ's on expression of several checkpoint proteins by western blot analysis. Treatment with CPFX at 20 or 40 µg/ml resulted in reduced protein expression of cdc2 (34 kDa) at five days, whereas reduced protein expression at 4 days was only apparent at the 40 µg/ml concentration (Figure 3). Consistent with results of the cell cycle analysis, while ENFX appeared less potent than CPFX, it was still effective at reducing the cdc2 expression in canine OSA cells; most notable at a concentration of 40 µg/ml on day 5 (Figure 3). In contrast, ENFX or CPFX resulted in similar increases in expression of phospho-cdc25C in canine OSA cells after 4 or 5 days of exposure.

Figure 3: G₂ checkpoint kinases affected by CPFX and ENFX treatment.

Western blot analysis of cdc2 (CDK1) and cdc25C in Abrams OSA cells at day 4 and 5. Both CPFX and ENFX inhibit G₂ checkpoint kinase cdc2 although CPFX showed more potent than ENFX. The level of phospho-cdc25C was increased in both CPFX and ENFX treated canine Abrams OSA cells.

Ciprofloxacin and enrofloxacin activate caspase- 3 and 7

Because OSA cell exposure to FQ's resulted in an increased sub G₁ fraction on cell cycle analysis, we wanted to examine whether FQ's were specifically capable of inducing apoptosis. We found that Caspase-3/7 activity increased significantly (p = 0.005, 0.001, 0.008, 0.002) in canine Abrams OSA cells after incubation either with 20 or 40 µg/ml of CPFX or ENFX (Figure 4A). CPFX induced activation of caspases was dose-dependent (p = 0.001), whereas no significant difference was detected between 40 µg/ml of ENFX and the 20 µg/ml concentration (p = 0.684).

Figure 4: Activation of apoptotic cell death induced by CPFX or ENFX.

A. Caspase-3/7 activity was measured in relative fluorescence unit with a commercial assay kit in canine Abrams OSA cells following incubation with CPFX (20 and 40 µg/ml) or ENFX (20 and 40 µg/ml) for 72 hours. ** Significantly (p<0.01) different from value for untreated control cells. B. Western blot analysis of PARP cleavage and cleaved caspase-3. Apoptosis is indicated by the increase of cleaved-PARP and cleaved-caspase-3.

Ciprofloxacin and enrofloxacin reduce expression of p21 ^WAF1

Lastly, to further investigate the mechanism of FQ induced changes in proliferation and survival we examined the effects of FQ's on p21 expression by Western blot. We found that expression of p21 in canine Abrams OSA cells was markedly reduced after 48 hour exposure to either CPFX or ENFX (Figure 5).

Figure 5: Decreased expression of p21 by CPFX or ENFX.

Western blot analysis in Abrams OSA cells treated with CPFX or ENFX at a dose of 20 µg/ml or 40 µg/ml at day 2. Abrams cells treated with doxorubicin were used as a positive control.

Discussion

While adjuvant chemotherapy is known to improve the disease-free interval and median survival time in canine and human patients treated with adequate surgical control, survival has remained relatively static for the past 20 years. However, post-operative limb-spare infections have been associated with improved survival in both species [3]–[5], [15]. While some evidence supports the notion that infection and/or a systemic anti-tumor immune response can inhibit growth of canine OSA [16]–[20], one clinical report noted that the majority of dogs experiencing post-operative infection were treated with FQ antibiotics [3]. We therefore questioned whether FQ's might have direct effects on canine OSA cells and set out to investigate any direct effects of commonly used FQ's on canine OSA cell lines in vitro.

ENFX is currently approved for use in several domestic species, and is commonly prescribed in veterinary species with susceptible infections. ENFX differs from CPFX by the presence of an ethyl group, but pharmacokinetic elimination of ENFX is partially accomplished by means of hepatic metabolism to CPFX [21], [22]. Canine tissue levels of ENFX and CPFX vary significantly between tissues and are dependent on the tissue type and the administered dose [23], [24]. In inflammatory tissues, the concentrations of ENFX and CPFX have been measured as high as 57 µg/g and 6 µg/g, respectively, after administration of a single 20 mg/kg dose of ENFX [24]. At this same administered dose, mean tissue concentrations of ENFX and CPFX after a single dose of ENFX reach 18.7 and 2.8 µg/g in the lung, respectively, [23] which are within the range of concentrations used in this study. However, it should be cautioned that these measured tissue concentrations are based on tissue homogenates and may not be truly representative of concentrations to which OSA cells would be exposed in vivo. We evaluated the effect of ENFX or CPFX in concentrations ranging from 1–40 µg/ml of the drug and found dose and time-dependent effects (Figure 1). We have further determined that similar levels of CPFX or ENFX can alter expression of cell cycle proteins and induce apoptosis of canine OSA cells.

Several studies have demonstrated the cytotoxic and apoptosis-inducing effects of FQ antibiotics on tendon cells, chondrocytes, and a variety of human cancer cell lines [8], [25]–[29]. However, previous studies used a wide range of concentrations, and multiple mechanisms of action have been implicated. The accumulation of Abrams cells treated with ENFX or CPFX in the S and G₂/M phase of the cell cycle with concomitant induction of apoptotic cell death is in agreement with previous reports of topoisomerase II inhibition by CPFX resulting in S-G₂/M arrest of prostate carcinoma, transitional cell carcinoma, and colorectal carcinoma cells [25], [28], [29]. While cell cycle analysis and apoptotic death were not specifically evaluated in one previous study using human OSA cells, our findings are consistent with previous reported results demonstrating that CPFX is capable of inhibiting the growth of human OSA cells at concentrations as low as 20 µg/ml [6].

Furthermore, we found that CPFX and ENFX decreased cdc2 expression concomitant with an increase in expression of phospho-cdc25C and directly determined an S-G₂/M cell cycle arrest by flow cytometry (Figure 2, ,3).3). Decreased expression of cyclin-dependent kinase 1 (cdc2) has previously been reported in rat tendon cells treated with 50 µg/ml of CPFX [30]. Regulation of cdc2-B1 complex involves and activating phosphate by CDK-activating enzyme and inhibitory phosphates at a pair of amino acids in the roof of the active site by Wee 1. Dephosphorylation of these sites by phosphatase cdc25C results in increased CDK activity [31]. DNA damage is associated with many cellular events, including activation of CHK1, which in turn phosphorylates and inactivates cdc25C, allowing inactivation of the 2-B1 complex and G₂/M arrest [32].

CPFX and ENFX also led to decreased expression of the cyclin dependent kinase inhibitor p21^WAF1 (Figure 5). Previous studies have also demonstrated p21^WAF1 down-regulation in CPFX treated cancer cells and indicated that the observed down-regulation of p21^WAF1 was likely due to the degradation of p21^WAF1 following ubiquitination by the 26S proteasome-mediated degradation pathway [28]; and that the observed degradation was closely correlated with the time of induction of apoptotic cell death [29]. Inhibition of p21^WAF1 has previously been shown to sensitize ME-180 OSA cells and MG63 human OSA cells during TNF or anti-Fas-induced apoptosis [33], [34]. This strongly suggests the need to further evaluate the effect of FQs on mitochondria as well as the proteasome-ubiquitination system in canine cancer cells and raises the possibility that FQ's could possibly enhance host mediated immunologic cytotoxicity of OSA.

In addition to cell cycle perturbations, our experiments revealed an in vitro apoptotic effect of CPFX or ENFX on canine OSA cells, in a dose - and time -dependent manner. Caspase-3 is a key member of the caspase family, which is the central component of the apoptotic machinery during apoptotic cell death. Poly (ADP-ribose) polymerase (PARP) is a common death substrate for activated enzymes of caspase family and caspase-3 and caspase-7 demonstrated both cleave PARP [35]. As shown in Figure 4B, activation of caspase-3 after ENFX or CPFX treatment of the canine OSA cells was confirmed by western blot analysis and the activation of caspase-3 was correlated with the proteolytic cleavage of PARP. These results correspond with previous investigations in human bladder cancer cells exposed to 100–300 µg/ml of CPFX [28].

The experiments in this study were designed to determine whether or not FQs were directly capable of inhibiting survival and proliferation in canine OSA cells. Based on our findings, it is possible that the use of FQ's in clinical patients may have contributed to the improved outcome of dogs with OSA. However, much work is needed before such a conclusion can be made. While our results support further study of FQs or possibly other quinolones in the treatment of OSA, many questions remain. Specifically of concern is that while reduction in p21 has been previously shown to increase apoptosis in some cells, loss of p21 expression can increase cellular proliferation in other cells and may contribute to carcinogenesis and tumor progression [36]. Therefore, our results must be interpreted with caution until further in vitro and in vivo studies can be performed.

Materials and Methods

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