Anti-echinococcal effect of verapamil involving regulating calcium/calmodulin-dependent protein kinase response in vitro and in a murine infection model

Hai-Jun Gao School of Basic Medical Sciences, Lanzhou University Xu-Dong Sun School of Basic Medical Sciences, Lanzhou University Yan-Ping Luo School of Basic Medical Sciences, Lanzhou University Hua-Sheng Pang National Health Commission Key Laboratory of Echinococcosis Prevention and Control, Xizang Center for Disease Control and Prevention Xing-Ming Ma School of Basic Medical Sciences, Lanzhou University Ting Zhang (  zhangting@nipd.chinacdc.cn ) National Institute of Parasitic Diseases, China CDC Tao Jing School of Basic Medical Sciences, Lanzhou Univeristy Wei Hu National Institute of Parasitic Disease, Chinese Center for Disease Control and Prevetion Yu-Juan Shen National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevetion Jian-Ping Cao National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevetion, WHO Collaborating Center for Tropical Diseases, National Center for International Research on Tropical Diseases, Key Laboratory of Parasitie and Vector Biology of the Chinese Ministry of Health

. According to the previous data of central Asia, at least 270 million people are being exposed to Echinococcus, and its prevalence in some Tibetan area of western China ranged as high as from 0.8-11.9% [3]. More notably, a higher pathogenicity and case fatality rate were associated with AE due to its tumor-like growth property [3]. The clinical treatment strategies associated with echinococcosis include surgical operation and drug chemotherapies. Among the chemotherapeutic agents, albendazole (ABZ), one of the benzimidazole derivatives, is the prime chemotherapeutic drug used for treating human echinococcosis [4], and exerts an anti-parasitic e cacy by disrupting the microtubule polymerization and biochemical processes, such as glucose and energy metabolism of the parasite [5]. However, ABZ merely exerts the parasitostatic rather than parasitocidical effects [6], and has poor gastrointestinal absorption and severe side effects [7,8]. Hence, new drugs against this parasitosis have been investigated, such as traditional Chinese medicine from some botanical extracts [9], antineoplastic chemotherapeutics [7,10], immunosuppressants [11,12] and so on. However, it is disappointing that only a few of these agents, such as me oquine or amphotericin B alone or combined with nitazoxanide, were used in auxiliary treatment for human echinococcosis [6]. Therefore, there is an urgent need to explore novel anti-Echinococcus drugs and drug-targets.
Ca 2+ , a pivotal second messenger, controls the physiological process of cell, such as the proliferation, differentiation and migration [13]. The complex of Ca 2+ and calmodulin (CaM) can speci cally bind to CaM-dependent protein kinases (CamKs, including CamK , CamK , CamK and CamK ) to construct the Ca 2+ /CaM-CamKs cascade that can turn on the signal transduction process of cells [14]. Previous studies in tumors have shown that Ca 2+ /CaM-CamKs cascade controls tumourigenesis and tumor progression [13,15,16]. Furthermore, Ca 2+ /CaM-CamKs cascade has been proved to be closely related to the pathogenesis of many hepatic parasites. For example, in Schistosoma mansoni, an IQ-motif of SmCav1B, which is a voltage-gated calcium channel, can interact with two calmodulins (CaMs) SmCaM1 and SmCaM2 for promoting the growth process [17], and RNAi silencing of calcium-regulated protein affects the morphology and vitality of Schistosoma japonicum [18]. Two voltage-gated calcium channel βsubunits, CsCa v β1 and CsCa v β2, have boosted the parasiticidal effect of praziquantel on Clonorchis sinensis [19], and as well, in Fascida hepatica, FhCaMs dyshomeostasis could obviously block its growth and motility [20]. Therefore, Ca 2+ /CaM-CamKs has become a potential therapeutic target in cancers and parasitosis, and calcium channel inhibitors, such as verapamil and praziquantel, were con rmed to exhibit anti-tumorigenic and anti-parasitic effects [13,15,21,22]. Further, verapamil has been shown to alleviate atrial brillation in rats by down-regulating the over-activation of Cav1.2-CaM-CaMK [23]. In E. granulosus PSCx, calcareous corpuscles have supported the necessity of calcium sources for hydatid cyst development [24], and calcium level in hydatid cyst is shown to be higher than that in the serum or plasma of host [25]. Old documents showed that verapamil is preliminary con rmed to suppress the growth of E. granulosus larva [26,27], but it is unclear if the compound also had similar anti-parasitic functions on E. multilocularis that is a tumor-like and more harmful parasite to humans. The mechanism of verapamil against Echinococcus has never been investigated, and it is unclear if the inhibitory effects of verapamil on E. granulosus should be processed by regulating Ca 2+ /CaM-CamK . Hence, this study aimed to investigate the e cacy and the possible mechanism of verapamil against Echinococcus.
Separation and culture of E. granulosus PSCx in vitro E. granulosus PSCx were obtained from naturally infected sheep liver in the slaughterhouse of Xining City, Qinghai Province, China, and then were rinsed with phosphate buffered saline (PBS) and resuspended with Dulbecco's modi ed Eagle's medium (DMEM) containing 1% penicillinan-streptomycin (P-S), and then incubated in 24-well culture plates (100 PSCx per well) at 37 °C, 5% CO 2 . The morphological alterations of all PSCx were observed under an inverted microscope (BX43, Olympus, Japan), and the survival rate of PSCx was recorded daily.
Mouse infected with E. multilocularis All experimental Kunming mice aged 6-8 weeks were purchased from the Laboratory Animal Center of Lanzhou University and maintained in a HEPA-ltered and temperature-controlled light/dark cycle environment at 22-25 ºC. The mice were fed with rodent diet (Beijing Keao, Beijing, China) ad libitum in speci c pathogen-free (SPF) laboratory conditions. E. multilocularis PSCx were aseptically isolated from the anesthetized E. multilocularis infected gerbil in our lab as described previously [28], with purpose to establish the murine infection model by in situ surgical intrahepatic implantation. The healthy mice (n = 15) were infected with E. multilocularis PSCx (1500 PSCx per mouse) in SPF lab, and other mice (n = 5) merely with a sham operation, i.e., the uninfected group, were raised under same conditions.

Drug treatment in vitro
The experimental groups were divided into (i) the vehicle group with 0.1% DMSO (n = 3), (ii) the ABZ-SO group with 40 and 20 µg/mL, wherein the ABZ-SO was resolved in 0.1% DMSO (n = 3), and (iii) the verapamil group with different concentrations of 100, 80, 40, 20, 10, 5, 2, 1 and 0.5 µg/mL (n = 3). E. granulosus PSCx on post-treatment with drugs were stained with 0.4% trypan blue for 10 min to observe the morphological changes. In addition, other E. granulosus PSCx were xed with 4% glutaraldehyde, rinsed with PBS (1×), stained with 2% osmium tetroxide for 2 hours and 1% uranyl acetate for 30 min. Subsequently, these specimens were dehydrated serially by increasing the concentrations of ethanol, dried naturally and then coated with gold as described [28]. Finally, the microstructure of PSCx was observed under a scanning electron microscope (SEM, JSM-5600LV, JEOL, Japan).

Drug treatment in vivo
On post-infection of E. multilocularis PSCx for 3 months, all E. multilocularis infected mice were grouped for orally administered drug therapy: (i) the infected mice were treated daily with only honey/PBS (1:1 v/v) (n = 5), (ii) ABZ mice were treated daily with 40 mg/kg ABZ in honey/PBS (1:1 v/v) (n = 5), (iii) verapamil mice were treated daily with 40 mg/kg verapamil in honey/PBS (1:1 v/v) (n = 5), and (iv) the uninfected mice were merely treated daily with honey/PBS (1:1 v/v) (n = 5). After 4 months posttreatment, E. multilocularis cysts, serum and liver were collected from the mice for testing the calcium content, and protein and mRNA expression of CaM and CamK genes.
Calcium content analysis by ICP-MS, SEM-EDS and alizarin red staining i. Inductively coupled plasma mass spectrometry (ICP-analysis: Equal tissues or serum/culture supernatant were prepared for calcium Tissue digestion was done in a microwave digestion system using UltraClave (Milestone, Sorisole, and the sample detection was processed as described previously [29]. ii. Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-Analysis: The changes in calcium content in the E. granulosus PSCx and in germinal layer cells of E. multilocularis metacestodes after treatment of verapamil were observed by a LEO Gemini Field Emission Gun-Scanning Electron Microscope (FEG-SEM, JEOL, Japan) as described previously [30].
iii. Alizarin red staining analysis: E. multilocularis metacestodes and mice liver were xed with 4% paraformaldehyde for 2 weeks, and then stained with alizarin red to detect the calci cation as described previously [31]. The images were analyzed by using Image J software (National Institutes of Health, Bethesda, MD, USA) to calculate the percentage of positively stained calcium deposits in the image eld.
CaM and CamK protein expression analysis by IHC-P i. Para n-immunohistochemistry (IHC-tests: 4 µm thick sections of E. multilocularis metacestodes and mouse liver were processed for evaluating CaM or CamK expression as described previously [32,33], followed by immunoreaction using rabbit anti-CaM/CamK antibody (Bioss, Beijing, China) at 1:300/1:200 and secondary antibody (Goat anti-rabbit IgG, Bioss, Beijing, China) at 1:800. Finally, the slides were viewed under a uorescence microscope (Olympus, Japan). The semi-quanti ed analysis was evaluated by using Image J software.
ii. ELISA tests: The serum and tissue samples were added to PBS (pH = 7.containing PMSF (10 mM/L), followed by rapid homogenization and centrifugation at 1000 ⋅ g for 10 min to investigate the concentrations of CaM and CamK proteins according to the ELISA kit protocols.
CaM and CamK mRNA analysis by RT-qPCR The expression of CaM or/and CamK mRNA in E. granulosus PSCx, mice liver and E. multilocularis cysts were tested by real-time quantitative polymerase chain reaction (RT-qPCR), and β-actin mRNA was used as an internal standard. Nucleic acid in different tissues was isolated by using Trizol reagent (Invitrogen, San Diego, USA), and then reverse-transcribed to cDNA, and ampli cation of cDNA was performed by RT-qPCR as described by Takara kit protocols (No. RR036A). The primers used were as follow: Eg-CamK /Em-Camk (forward, 5'-TCGTTGTTCAAGTCGGTTCG-3'; reverse, 5'-GGTGCTGAGA

Statistical analysis
The data were presented as means ± standard deviation (SD). Statistical differences among different groups were assessed by T test and paired comparisons. Statistical analysis was performed by SPSS version 22.0 (IBM Corp., Chicago, USA) and GraphPad Prism version 7.0 (GraphPad Software, Inc., San Diego, CA, USA). P < 0.05 indicated statistical signi cant differences.

Effect of verapamil on E. granulosus PSCx in vitro
The survival rate of E. granulosus PSCx after treatment with a series of concentrations of verapamil at 0.5, 1, 2, 5, 10, 20, 40, 80 and 100 µg/mL within 8 days was shown in Fig. 1A. The mortality rate of PSCx showed a time-dependent property when exposed to 0.5-40 µg/mL verapamil, and all PSCx were killed within 2 days at 40 µg/mL or 4 days at 20 µg/mL. However, ABZ-SO merely killed 13% PSCx on day 2 at 40 µg/mL or 21% on day 4 at 20 µg/mL, and 4% PSCx were dead on day 2 or 8% on day 4 in the vehicle group.
The results of light microscope showed when compared to the natural morphology of PSCx in the vehicle group and mild alterations of PSCx in ABZ-SO (20 µg/mL) group, an obvious morphological alterations (i.e. disappearance of calcareous corpuscles) of PSCx was observed after exposed to verapamil 20 µg/mL for 4 days, (Fig. 1Ba-c). In addition, PSCx in verapamil (20 µg/mL) group on day 4 was dyed with trypan blue, unlike the viable PSCx in ABZ-SO (20 µg/mL) group or vehicle group (Fig. 1Bd-f). The ultrastructural destructure in PSCx, involving the shedding of the tegument, the disappearance of the hooks and the presence of numerous blebs, were observed by SEM when exposed to verapamil (20 µg/mL) for 4 days, while that in the vehicle group or in ABZ-SO (20 µg/mL) group were still shown to be intact (Fig. 1Bg-i).
Changes of calcium content in E. granulosus PSCx exposed to verapamil in vitro The calcium distribution in E. granulosus PSCx when treated with verapamil (20 µg/mL) for 4 days has become heterogeneous and sparse as compared with that in the vehicle group by SEM-EDS ( Fig. 2A). The semi-quantitative analysis showed that the calcium level in PSCx was decreased after treatment of verapamil (Fig. 2B). In addition, compared with the vehicle group, an obvious drug-dose dependent increase of the calcium content in the culture supernatant of the verapamil group was observed (Fig. 2C).
Effect of verapamil on E. multilocularis metacestodes in vivo E. multilocularis infected mice were treated with verapamil (40 mg/kg) or ABZ (40 mg/kg) for 4 months in vivo. The wet weight of E. multilocularis cysts from the verapamil (0.98 ± 0.33 g) or ABZ group (1.04 ± 0.14 g) showed signi cant declination when compared with that from the infected group (5.90 ± 0.75 g) (P = 0.000, Table 1).

Changes of calcium concentration in E. multilocularis infected mice after verapamil treatment
The calcium concentration of serum in E. multilocularis infected mice was 4.92 ± 0.77 mg/L, which was declined by 2-folds when compared with the uninfected group (9.89 ± 1.92 mg/L), while post-treatment with verapamil for 4 months showed the recovery of calcium concentration to 6.39 ± 0.79 mg/L (Table 2). Similarly, the level of calcium in the liver of mice was declined from 296.72 ± 9.43 mg/L to 172.72 ± 16.63 mg/L due to E. multilocularis metacestodes infection. Interestingly, the calcium content in the liver was increased to 226.78 ± 43.93 mg/L after treatment of verapamil (P = 0.009). Additionally, ICP-MS assay showed that the calcium level in E. multilocularis cysts from the infected group was 3182. 28 ± 190.77 mg/L, which was merely decreased to 3013.98 ± 115.80 mg/L after treatment of verapamil for 4 months (P = 0.13). Furthermore, calcium changes in E. multilocularis cysts were evaluated by SEM-EDS (Fig. 3A), and the percentage of calcium weight in E. multilocularis cysts demonstrated a swift drop from 14.28-8.66% after treatment with verapamil, showing statistical signi cant differences (P = 0.000) (Fig. 3B). Alizarin red staining showed that calcium deposition around the portal area of the liver in the infected mice was increased signi cantly when compared with that in the uninfected group (Fig. 4). The loss of calcium content in the infected livers showed no apparent recovery after treatment of verapamil, and meanwhile in E. multilocularis cysts, no obvious reduce of calcium content was observed (P > 0.05), but the reduce in the number of PSCx was observed. Analysis of CaM and CamK protein in E. multilocularis infected mice treated with verapamil IHC-P staining exhibited high expression of CaM in the liver of the infected group when compared with that in the uninfected group (Fig. 5A), but the pathological progression could be inhibited by verapamil, showing statistical signi cant differences (P < 0.05). Furthermore, ELISA assay showed that the CaM protein concentration in mouse serum or liver increased signi cantly from 13.81 ± 1.65 to 22.25 ± 5.55 µg/mL or from 3.42 ± 0.27 to 6.06 ± 1.83 µg/mL after E. multilocularis infection, but after treatment with verapamil, an obvious inhibitory effect on CaM protein expression was observed in the serum (8.13 ± 1.26 µg/mL) and liver (1.60 ± 0.68 µg/mL) not in the cysts from 2.36 ± 0.87 to 1.68 ± 0.10 µg/mL (Table 3). Similar to CaM, IHC-P assay supported that the over-expression of CamK of the liver and cysts in E. multilocularis infected mice were signi cantly suppressed by verapamil treatment (Fig. 5B). In addition, the increase of CamK content in mice serum (22.87 ± 4.23 ng/mL) and liver (4.25 ± 1.84 ng/mL) were observed after E. multilocularis infection, and then were decreased to 5.13 ± 1.74 ng/mL and 1.97 ± 0.56 ng/mL after treatment of verapamil. However, only a mild decrease of CaMK content in cysts (from 2.65 ± 1.24 to 1.79 ± 0.36 ng/mL) was observed after verapamil treatment (P > 0.05, Table 3).
Analysis of CaM and CamK mRNA in parasite and E. multilocularis infected mice after treatment with verapamil To further observe the change of Ca 2+ /CaM-CamK in parasite infected mice after treatment of verapamil, the change in CaM or/and CamK mRNA of mice liver and Echinococcus were evaluated by using RT-qPCR. Figure 6A-B showed that CaM and CamK mRNA expression in mice liver obviously increased by 4-fold and 6-fold after infection of Echinococcus. Nonetheless, the over-expression of CaM and CamK mRNA in the liver were signi cantly suppressed by verapamil (P = 0.000). Meanwhile, in the cysts after treatment of verapamil, obvious down-regulation was observed in CamK mRNA expression not in CaM mRNA expression with mild reduce (P > 0.05). Furthermore, in E. granulosus PSCx exposed to verapamil in vitro, obvious down-regulation of CamK mRNA expression was observed (Fig. 6C).

Discussion
E. granulosus and E. multilocularis metacestodes are inclined to be parasite on the liver, and E. multilocularis metacestodes with tumor-like growth property has become more noticeable and leads to death if untreated [34]. While accompanied with poor intestinal absorption and heavy side effects [7], ABZ, exerting parasitostatic rather than parasitocidical effect, has long been used for treating for human echinococcosis. Hence, the urgency of exploring new anti-echinococcal targets and therapeutic options should be considered.
Ca 2+ /CaM-CamKs as a potential therapeutic target in cancers and many parasitosis is closely monitored, but it is unclear as to how Ca 2+ /CaM-CamK regulates the growth and development of Echinococcus spp. and requires urgent exploration with the purpose to nd potential drugs for treating echinococcosis at early stages.
In this study, the anti-echinococcal effect of verapamil on E. granulosus PSCx was found to be timedependent and dose-dependent, which was also observed when studying the anti-tumor proliferation. As is well-known, calcareous corpuscles in PSCx can persistently provide abundant calcium sources to promote PSCx development into cysts [24]. In the presented study, the rapid disappearance of calcareous corpuscles and the loss of calcium content in E. granulosus PSCx were observed when exposed to verapamil, indicating that verapamil could kill E. granulosus PSCx by promoting calcium loss.
AE is usually named a "parasitic cancer" due to its distinctive tumor-like growth properties, and approximately 70% of the metacestodes were found in the right lobe of the patient's liver. The germinal layer cells in E. multilocularis metacestodes with high regenerative capacity can develop into new multicular structures, such as PSCx, which can further develop into new metacestodes again. So, the E. multilocularis metacestodes can in ltrate the whole liver of host [35]. In this study, the infection model was established by in situ surgical intrahepatic implant to follow the natural occurrence and development of AE in the mouse as possible. After E. multilocularis infected mice were treated with verapamil 40 mg/kg for 4 months, the weight of E. multilocularis cysts was decreased signi cantly, suggesting that the growth of metacestodes was suppressed. This was similar to the results of Cao's observation of verapamil on CE mice [26]. It was reported that the infection of E. multilocularis metacestodes reduced serous calcium levels in both mice and humans [25,36]. Interestingly, our results discovered that the reduction of calcium content in E. multilocularis infected mice was signi cantly recovered under administration of verapamil, and the apparent suppression was observed in liver rather than in serum or cysts with mild increase. The calcium content in E. multilocularis metacestodes is much higher than that in the host liver and serum, indicating that E. multilocularis PSCx development into metacestodes needs continuous absorption of calcium from the host. Furthermore, the results of SEM-EDS assay showed that after treatment with verapamil, both the calcium content and number of germinal layer cells were obviously reduced. Thus, we speculated that the proliferation of germinal layer cells was regulated and controlled by calcium supply in infected mice and can be broken by calcium channel inhibitor verapamil. However, the detailed mechanism of suppressing the growth of germinal layer cells by verapamil regulating Ca 2+ /CaM-CamKs cascade needs to be further explored. Further, alizarin red staining and ICP-MS results also supported that E. multilocularis metacestodes could cause calcium content translocation from mice into the parasite, and calcium channel inhibitor (verapamil) would rescue the calcium loss of E. multilocularis infected mice. In addition, abundant lymphocytes were distributed in the liver around E. multilocularis cysts after treatment with verapamil, which is potentially associated with the increase of calcium content in the liver of E. multilocularis infected mice after treatment with verapamil, the probable reason is that enough calcium could promote the proliferation and polarization of T cells [37], but the abnormality of lymphocytes development caused by loss of calcium in Echinococcus infected host still requires further exploration.
We found that the increased expression of CaM and CamK protein in mouse serum and liver were caused by E. multilocularis metacestodes infection, but the over-expression of CaM and CamK protein were suppressed signi cantly by verapamil. Furthermore, the expression of CaM and CamK mRNA in mice liver and E. multilocularis metacestodes were down-regulated after treatment with verapamil. Our results partially supported Sujeevi S. K. Nawaratna's recent ndings that Ca 2+ /CaM-CamK in helminth parasite are putative therapeutic targets that could provide biochemical and pharmacological information for exploring novel compounds in the future [38]. Ca 2+ /CaM-CamK controls the completion of life cycle in Echinococcus, and calcium channel blocker verapamil exhibits strong inhibitory effects on Echinococcus, including the germinal layer cell, PSCx and metacestodes. Therefore, further study should emphasize on the molecular and immune mechanisms of verapamil on the over-activated Ca 2+ /CaM-CamK in Echinococcus infected host. On the other hand, based on Ca 2+ /CaM-CamK , seeking novel anti-echinococcal drug-targets and effective treatment strategies should be carefully investigated.

Conclusions
Our study results suggested that verapamil had parasiticidal e cacy on both E. granulosus PSCx in vitro by promoting calcium loss, and E. multilocularis metacestodes in vivo by inhibiting calcium content translocating from mice to parasite. Furthermore, Ca 2+ /CaM-CamK in the mouse was over-activated by Echinococcus infection, which was alleviated after oral administration of verapamil. Thus, our ndings CamK , Ca 2+ /calmodulin dependent protein kinase ; ABZ-SO, albendazole sulfoxide; IHC-P, immunohistochemistry-para n; PBS, phosphate buffered saline; SEM-EDS, scanning electron microscope and energy dispersive spectrometer; ICP-MS, inductively coupled plasma mass spectrometry; RT-qPCR, real-time quantitative polymerase chain reaction.

Declarations
Ethics approval and consent to participate This study was conducted in accordance with the Chinese Laboratory Animal Administration Act and the study protocol was approved by the Experimental Animal Ethics Committee of School of Basic Medical Sciences, Lanzhou University (permit no. 2014-12-003).

Consent for publication
Not applicable.

Availability of data and materials
The Datasets supporting the conclusions of this article are included within the article.
Competing interests