Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice

Background HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics. Methods Cytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models. Results Zn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin. Conclusion Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.

of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin.
Conclusion: Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.

Background
Heme oxygenase (HO) is a microsomal enzyme that catalyzes oxidative cleavage of the porphyrin ring in heme molecule leading to the formation of biliverdin, carbon monoxide (CO) and free iron [1,2]. Biliverdin is further converted into bilirubin by biliverdin reductase. All HO products exert pleiotropic effects including numerous cytoprotective responses [3]. Bilirubin and biliverdin are among the most potent endogenous scavengers of reactive oxygen species (ROS) [4]. CO exerts strong antiapoptotic and anti-inflammatory effects through induction of soluble guanylyl cyclase. It suppresses production of tumor necrosis factor (TNF), interleukin-1β (IL-1β) and CCL4 chemokine (macrophage inflammatory protein-1β), but up-regulates synthesis of anti-inflammatory IL-10 [5]. Finally, free iron (Fe 2+ ) despite participation in Fenton reaction that leads to formation of highly reactive hydroxyl radicals, also activates Fe-ATPase, a transporter that removes intracellular iron, as well as induces expression of ferritin heavy chains which sequester free iron and exert specific cytoprotective roles [6].
Two isoforms of heme oxygenase exist. HO-1 is an inducible enzyme that belongs to the heat shock protein (HSP32) family. Its expression is induced by a vast array of stress-inducing stimuli that include: oxidative stress, heat shock, UV irradiation, exposure to heavy metals and numerous other toxins, including chemotherapeutics [7]. Some observations indicate that HO-1 and its products also exert anti-inflammatory effects and participate in the control of growth and proliferation of tumor cells. Elevated constitutive levels of HO-1 have been observed in a number of human tumors including glioma, melanoma, prostate, pancreatic and renal cell carcinoma, lymphosarcomas, Kaposi sarcoma and hepatoma [7]. Enhanced expression of HO-1 can also contribute to tumor progression through promotion of angiogenesis and metastases formation [8,9]. Furthermore, the increased basal level of HO-1 expression in tumor cells can be further elevated by chemotherapeutics, radiotherapy or photodynamic therapy [10,11].
Altogether HO products participate in attenuation of oxidative stress, suppression of inflammatory responses, inhibition of apoptosis and promotion of angiogenesis [12,13]. Therefore, accumulating evidence indicates that HO-1 can be a therapeutic target for antitumor treatment.
Indeed, it was shown that zinc protoporphyrin IX (Zn(II)PPIX) or its pegylated derivative, a potent HO inhibitor, can exert significant antitumor effects against several tumors in mice [14][15][16]. Moreover, inhibition of HO-1 expression or activity was shown to increase responsiveness of tumor cells to other anticancer treatments in vitro [10,16,17]. The aim of these studies was to explore the in vivo role of HO-1 in tumor growth and in protecting tumor cells against chemotherapeutics.

Reagents
Zinc (II) propoporphyrin IX (Zn(II)PPIX), a HO-1 inhibitor, was purchased from Frontier Scientific Europe Ltd. (Carnforth, Lancashire, United Kingdom) and was dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, USA) to the final stock concentration of 5 mM. Bilirubin (Sigma, St Louis, MO, USA) was dissolved in 0.1 N NaOH to the final stock concentration of 10 mM. Hemin (from Sigma) was dissolved in 0.1 N KOH to the final stock concentration of 10 mM. All the solutions were prepared in the dark right before adding to the cell cultures.

Mice
Female BALB/c and C57Bl/6 mice, 8-12 weeks of age were used for in vivo experiments. Breeding pairs were obtained from the Institute of Oncology (Warsaw, Poland). Mice were kept in conventional conditions with full access to food and water during experiments. All of the animal studies were performed in accordance with the guidelines approved by the Ethical Committee of the Medical University of Warsaw.

Cytostatic/cytotoxic assay
The cytostatic and/or cytotoxic effects were measured using crystal violet staining as described [18]. Briefly, tumor cells were dispensed into 96-well plates (Sarstedt, Numbrecht, Germany) at a concentration of 5 × 10 3 cells per well and allowed to attach overnight. The following day investigated agents were added at indicated concentrations. Cells were kept in dark for 48 or 72 h. After the incubation time the cells were rinsed with PBS and stained with 0.5% crystal violet in 2% ethanol for 10 min at room temperature. Plates were washed four times with tap water and the cells were lysed with 1% SDS solution. Absorbance was measured at 595 nm using an enzyme-linked immunosorbent assay reader (SLT Labinstrument GmbH, Salzburg, Austria), equipped with a 595 nm filter. Cytotoxicity was expressed as relative viability of tumor cells (% of control cultures incubated with medium only) and was calculated as follows: relative viability = (A e -A b ) × 100/(A c -A b ), where A b is the background absorbance, A e is experimental absorbance, and A c is the absorbance of untreated controls.

Clonogenic assay
MDAH2774 or Mia PaCa2 cells were plated at 2.5 × 10 5 cells per 35-mm dish (Sarstedt). Four hours after seeding, Zn(II)PPIX was added at indicated concentrations. After 24 hours of incubation in dark, the cells were washed with PBS, trypsinized and seeded into 35-well plates in triplets at the concentration of 1 × 10 3 cells per a dish. Fresh medium containing Zn(II)PPIX was added. The medium was removed daily for the 6 following days. After 14 day incubation in the dark, the cells were rinsed with PBS, fixed for 10 min in pure methanol and stained with 0.5% crystal violet in 2% ethanol for 10 min at room temperature. Then the plates were washed four times with tap water and air-dried. The images of the plates were made using the Olympus Camedia C750 Ultra Zoom digital camera.

FACS analysis of cell cycle and apoptosis
For the cell cycle analysis, C-26 cells were seeded into 6well plates (Sarstedt) at the concentrations 1 to 3 × 10 5 cells per well and incubated in dark with 2.5 μM or 5 μM Zn(II)PPIX. The incubation time varied from 24 to 72 h. After the incubation, the cells were washed with PBS, trypsinized and centrifuged at 1000 rpm for 10 min in 4°C. The pellet was resuspended in 0.5 ml of PBS and injected under the surface of ice cold 70% ethanol for 24h fixation in -20°C. At the day of analysis, the cells were washed from ethanol, stained with 5 μg/ml propidium iodide (PI, Becton Dickinson, Mountain View, CA, USA) at the presence of RNase A (Becton Dickinson) for 30 min at 37°C. For the analysis of apoptosis induction C-26 or Mia PaCa2 cells were seeded into 6-well plates (Sarstedt) at the concentrations 1 to 3 × 10 5 cells per well and incubated in dark with 2.5 μM or 5 μM Zn(II)PPIX. The incubation time varied from 6 to 72 h. After the incubation, the cells were washed with PBS, trypsinized and centrifuged at 1000 rpm for 10 min in 4°C. The pellet was resuspended in 0.5 ml of Annexin-binding buffer (BioSource International, Camarillo, CA, USA) and then incubated for 15 min with 5 μl of Annexin V-FITC (BioSource). Finally, the cells were stained with 5 μg/ml PI. The cytofluorometric analysis was performed using FACSCalibur (Becton Dickinson). For single analysis 1 × 10 4 cells were used. Data were collected at the wavelength of 580 nm (for PI) and 520 nm for FITC, and analysed with CEL-LQuest 1.2 software (Becton Dickinson).

HO-1 RNAi
Small interfering RNA against murine HO-1 was obtained by chemical synthesis from Dharmacon (Lafayette, CO, USA). The following sequence was used for targeting of murine HO-1: 5'-GCA-GAA-CCC-AGU-CUA-UGC-C-3'. For RNAi experiments 3,5 × 10 5 C-26 cells were seeded into 6-well plates (Sarstedt). After 24 h cells were washed with Optimem™ medium (Invitrogen) and transfected with 100 nM siRNA using OligofectAMINE™ according to the manufacturer's protocol. 10 μM hemin was added to the appropriate groups 2 h befor RNAi procedure. After 8 h trasfection medium was replaced with full DMEM culture medium. 24 h after transfection cells were harvested and ROS generation analysis was performed (see below).

ROS generation assay
For determination of ROS generation, C-26 cells (3,5 × 10 5 cells per well) were seeded into 6-well paltes (Sarstedt) and treated for 24 h with 2.5 μM Zn(II)PPIX and/or 50 μM biliubin or subjected to HO-1 RNAi (as described above). On the day of the analysis, cells were trypsynized, washed 3 times in ice-cold PBS and resuspended in 1 ml of PBS. One μl of CM-H 2 DCFDA (Invitrogen, 5 mM DMSO solution) was added to each sample for 20 min incubation at 37°C. Then, cells were washed twice with ice-cold PBS and subjected to flow cytometry using FACSCalibur (Becton Dickinson). For single analysis 1 × 10 4 cells were used. Data were collected at the wavelength of 520 nm and analysed with CELLQuest 1.2 software (Becton Dickinson).

Tumor treatment and monitoring
For assessment of antitumor activity of Zn(II)PPIX in vivo, exponentially growing C-26 were harvested, re-suspended in PBS medium to the appropriate concentration, and injected at the dose of 1 × 10 5 cells per mouse into the footpad of the right hind limb of experimental mice. Tumor cell viability measured by trypan blue exclusion was always above 95%. For in vivo treatment Zn(II)PPIX was dissolved in DMSO and further diluted in 0.9% NaCl to required concentrations. Final DMSO concentration was always less then 0.1%. Zn(II)PPIX was distributed intraperitoneally at doses from 12.5 to 50 mg per kg of body weight or orally at doses from 11 to 22 mg per kg of body weight. Control animals received 0.1% DMSO solution in 0.9% NaCl i.p. or orally.
For in vivo experiments evaluating the effectiveness of combine treatment using Zn(II)PPIX and chemotherapeutics, exponentially growing C-26, EMT6 and B16F10 cells were injected at the dose of 1 × 10 5 , 1 × 10 5 and 1 × 10 6 cells per mouse, respectively into the footpad of the right hind limb of experimental mice. Zn(II)PPIX treatment (50 mg/kg i.p.) was started on the day 7 after inoculation of tumor cells and continued for 7 consecutive days. First dose of HO-1 inhibitor was administered 1 h before each of the chemotherapeutics to eliminate any possible interactions (such as neutralization) between drugs. Cisplatin (Platidam, Pliva-Lachema, Cech Republic) at the dose of 7.5 mg/kg i.p. In all the experiments mentioned above, local tumor growth was determined every second day as described previously [19] by the formula: tumor volume (mm 3 ) = (longer diameter) × (shorter diameter) 2 .

Statistical analysis
Data were calculated using Microsoft™ Excel 2003. Differences in in vitro cytotoxicity assays and tumor volume were analyzed for significance by Student's t test. Significance was defined as a two-sided P < 0.03.

Zn(II)PPIX induces potent cytostatic/cytotoxic effects against murine and human tumor cells
Four different cell lines of murine (C-26, colon adenocarcinoma) and human (Mia PaCa2, a pancreatic cancer, MDAH2774, ovarian carcinoma, and MDA-MB231, breast carcinoma) origin were incubated with increasing concentrations of Zn(II)PPIX for 48 and/or 72 hours. HO-1 inhibitor exerted dose-and time-dependent cytostatic/ cytotoxic effects as measured with crystal violet staining (Fig. 1). Similar effects were observed in MTT assay (not shown) and in a clonogenic assay performed with Mia PaCa2 and MDAH2774 cells (Fig. 2).
To get insight into the mechanism of cytostatic/cytotoxic effects induced by Zn(II)PPIX cell cycle analysis and induction of apoptosis were performed. Incubation of C-26 cells with Zn(II)PPIX for 48 or 72 h resulted in doseand time-dependent reduction of cells in G1 phase of the cell cycle (Fig. 3A). This effect correlated with decreased cyclin D1 expression (Fig. 3B) and increased percentage of cells in sub-G1 phase (Fig. 3A). Propidium iodide and annexin V staining of C-26 cells incubated with 5 μM Zn(II)PPIX for 24-72 h indicated that the sub-G1 fraction of cells might represent apoptotic cells (Fig. 3C). The fraction of late apoptotic and necrotic cells increased from 10.9% in controls to 30.4% after 72 h incubation with Zn(II)PPIX. Western blotting analysis indicated that a 48h incubation of C-26 cells leads to accumulation of cleaved (active) caspase-3 (Fig. 3D).
An influence of Zn(II)PPIX as well as knock-down of HO-1 expression with siRNA were used to get insight into the specificity of the Zn(II)PPIX-mediated effects. As shown in Fig. 3E siRNA against a murine HO-1 (moHO-1 siRNA) was effective in reducing the expression level of HO-1 in murine C-26 cells. A siRNA against a human gene (huHO-1 siRNA) was ineffective (Fig. 3E) as well as an irrelevant siRNA against enhanced green fluorescent protein (eGFP, not shown). Inhibition of HO-1 activity with Zn(II)PPIX resulted in increased ROS generation in C-26 cells (Fig.  3F). Similar effects were observed when HO-1 expression was knocked-down with a specific siRNA against HO-1 (Fig. 3G). The influence of Zn(II)PPIX on ROS formation was completely abrogated when C-26 were co-incubated with 50 μM bilirubin (Fig. 3F). Similarly, the influence of siRNA was significantly decreased by forced expression of HO-1 by pre-incubation of C-26 with 10 μM hemin (Fig.  3G).

Zn(II)PPIX induces antitumor effects in a murine C-26 model
BALB/c mice inoculated with C-26 cells were treated with Zn(II)PPIX for 7 consecutive days. HO-1 inhibitor was administered either intraperitoneally (i.p.) or per os and the tumor volume was monitored every second day, starting from day 7 after inoculation of tumor cells. Zn(II)PPIX exerted dose-dependent antitumor effects manifested by the retardation of tumor growth. A statistical significance was reached on days 17 and 19 for Zn(II)PPIX administered at a dose of 25 mg/kg either i.p. or orally (Fig. 4A and 4B). A stronger effect was observed when Zn(II)PPIX was administered i.p. at a dose of 50 mg/kg, where a statistically significant retardation of tumor growth was observed on days 13-19, as compared with controls (Fig. 4A). Figure 2 The influence of Zn(II)PPIX on formation of tumor cell colonies. For the clonogenic assay, MDAH2774 or Mia PaCa2 cells were plated at a concentration of 1 × 10 3 cells/dish. Medium containing Zn(II)PPIX was replaced daily for 1-6 consecutive days. On day 14 after PDT, the plates were fixed with methanol and stained with crystal violet.

Overexpression of HO-1 in B16F10 cells confers resistance to cisplatin treatment
B16F10 cells were transfected with HO-1 gene (B1 clones) or an empty control plasmid (B5E cells). Tumor cells were inoculated with mixtures of clones and their response to cisplatin treatment was investigated. Cisplatin was administered i.p. at three doses of 2.5, 5.0 or 7.5 mg/kg and the tumor growth was monitored every second day. While in control B5E tumors cisplatin administration led to a significant retardation of tumor growth, the B1 tumors were completely resistant to the chemotherapeutic (Fig. 5A and  5B). Administration of 50 mg/kg Zn(II)PIX alone produced significant retardation of tumor growth only in B5E tumor model (Fig. 5D), but was completely ineffective in B1 tumors (Fig. 5C). Furthermore, it did not restore cisplatin sensitivity in B1 tumors (Fig. 5C).

Zn(II)PPIX does not affect antitumor effects of chemotherapeutics
In further studies the influence of Zn(II)PPIX (50 mg/kg) on the in vivo antitumor effects of chemotherapeutics was evaluated. Three different cell lines syngeneic with BALB/ c or C57Bl/6 mice were used, namely C-26, B16F10 melanoma and EMT6 breast adenocarcinoma. The following chemotherapeutics were used in these studies: 5fluorouracil at a dose of 50 mg/kg (5-FU, for C-26), cispl-atin at a dose of 5 mg/kg (for B16F10) and doxorubicin at a dose of 7,5 mg/kg (for EMT6 cells). Although in in vivo studies administration of Zn(II)PPIX (at a dose of 50 mg/ kg) resulted in retardation of tumor growth (although in EMT6 tumors the effect was only modest) there was no further potentiation of the antitumor effects by concomitant administration of Zn(II)PPIX together with chemotherapeutics ( Fig. 6A-C). Only for Zn(II)PPIX and 5-FU a slightly stronger effect was observed for the combination treatment, but the difference between the combination and single drug-treated tumors did not reach statistical significance (Fig. 5A). Remarkably, the combined administration of Zn(II)PPIX with either cisplatin or 5-FU resulted in significant weight loss (Fig. 6D and 6E). This effects was not observed in mice treated with Zn(II)PPIX and doxorubicin (Fig. 6F). No treatment-related mortality was observed in these experiments.

Discussion
The role of HO-1 in tumor development is still not completely elucidated and some recent reports demonstrate discordant or even completely opposite results. Nevertheless, accumulating evidence indicates that HO-1 is expressed or overexpressed by a large variety of human tumors and that it plays a critical role in progression of neoplastic diseases [7]. For example, it was shown that increased expression of HO-1 is associated with higher proliferation rate of various tumor cells [7,10,15,20], although opposite effects are observed in breast cancer cells [21]. Specific siRNA-mediated down-regulation of HO-1 resulted in suppression of proliferation of pancre-atic cancer cells [10] or in induction of apoptosis of lung cancer cells [17]. Similarly, cytostatic/cytotoxic effects were observed with HO-1 inhibitors, such as Zn(II)PPIX [15,22] or its pegylated derivative (PEG-ZnPP) [16]. Moreover, overexpression of HO-1 increased viability, Antitumor effect of cisplatin or the combined treatment with Zn(II)PPIX and/or cisplatin in mice transplanted with control or HO-1 overexpressing B16F10 tumors proliferation and angiogenic potential of melanomas [9]. Mice inoculated with HO-1 overexpressing melanomas fared worse than controls, had a higher number of metastases and a significantly shortened survival [9].

Zn(II)PPIX exerts antitumor effects against C-26 adencarcinomas in mice
The results of present experiments performed with additional cell lines further establish that Zn(II)PPIX is an active agent capable of inducing cytostatic and cytotoxic effects against a number of human and mouse tumor cell lines. Moreover, in two different tumor models Zn(II)PPIX was demonstrated to exert potent antitumor effects manifested by the retardation of tumor growth.
Development of effective antitumor regimens requires administration of drugs in combination. The role of the combination treatment is to target different pathways in tumor cells in order to elicit more robust antitumor response. Synergistic antitumor effects might lead to decreased dosing and elimination or a significant attenuation of toxic side effects of drugs used in monotherapy. Several previous reports demonstrated that HO-1, which is frequently over-expressed in tumor cells, might participate in tumor-protective effects against a number of chemotherapeutics, radiotherapy and photodynamic therapy [10,11]. By targeting HO-1 it might be possible to sensitize tumor cells to more effective cytotoxic effects of other therapeutic regimens. Indeed, in vitro data seem to support this idea. HO-1 knock-down sensitized pancreatic cancer cells to gemcitabine [10] and lung cancer cells to cisplatin [17]. Moreover, PEG-ZnPP sensitized colon cancer cells to cytostatic/cytotoxic effects of camptothecin or doxorubicin [16]. However, the efficacy of combining chemotherapeutics with HO-1 inhibitor in vivo has not yet been addressed.
Antitumor effect of the combined treatment with Zn(II)PPIX and/or chemotherapeutics Considering the use of HO-1 inhibitors in combination with other antitumor agents it must be kept in mind that HO-1 and its products affect multiple signaling and metabolic pathways and play an important role in protection of normal cells against multiple environmental insults. For example, HO-1 protects retinal cells against lightinduced damage [23], ameliorates ischemia-reperfusion injury elicited by a number of different conditions [24], inhibits endothelial cell apoptosis induced by endoplasmic reticulum stress [25], protects mice from apoptotic liver damage [26], prevents development of atherosclerosis in LDL-receptor knock-out mice [27], improves survival of transplanted organs [28] or prevents development of gastric ulcers in rats [29]. HO-1 also protects normal cells and tissues against toxic effects of chemotherapeutics. Heme-induced HO-1 expression protects against cyclophosphamide-mediated hemorrhagic cystitis [30], HO-1 attenuates cisplatin-induced toxicity in renal tubular cells [31] or in auditory cells [32], it also decreases doxorubicin-mediated cardiotoxicity [33]. Moreover, transgenic mice deficient in HO-1 (-/-), develop more severe renal failure and have significantly greater renal injury compared with wild-type (+/+) mice treated with cisplatin [34].
At doses used in the studies presented here Zn(II)PPIX was unable to restore cisplatin sensitivity in HO-1 overexpressing melanoma cells nor was it capable of potentiating antitumor effects of cisplatin, doxorubicin or 5-FU in three different models. Nonetheless, Zn(II)PPIX significantly potentiated toxicity of cisplatin and 5-FU as determined by decreased weight loss. Tozer et al, have shown that administration of Zn(II)PPIX at a dose of 45 μmoles/ kg is ineffective in inhibiting HO-1 activity in tumors [35]. The dose used in these studies (50 mg/kg) corresponds to almost 80 μmoles/kg/mouse. Although it was not measured whether Zn(II)PPIX used at this dose and at this administration schedule was capable of inhibiting HO-1 activity it can be concluded that it is ineffective in neither inducing antitumor effects in HO-1-overexpressing B16F10 melanomas nor in restoring sensitivity to cisplatin. Future studies should address an important, and not addressed in these studies, question of drug administration schedule. Specifically, what should be the timing of Zn(II)PPIX administration in combination with chemotherapeutics to effectively inhibit HO-1 activity in tumor cells? Is it possible to design combination strategies that would target HO-1 in tumors and not in normal tissues? It seems that the influence of Zn(II)PPIX administration on the activity of HO-1 in tumor versus normal tissues will be of utmost importance in designing combination treatments.

Conclusion
Altogether, these studies show that despite promising cytostatic/cytotoxic and even antitumor effects elicited by Zn(II)PPIX, this HO-1 inhibitor should not be used in combination with chemotherapeutics. It can be hypothesized however, that selective and efficient delivery of HO-1 inhibitors to the tumor might prove to be a more rational approach for combination therapies with chemotherapeutics. Indeed, as demonstrated by Fang et al. [16] and Iyer at al. [36] it is possible to prepare Zn(II)PIX derivatives with a higher tumor selectivity. It remains to be elucidated whether these strategies are more effective and less toxic.