**8. Statins as adjuvant treatment in malignant hemopathies**

After chemotherapy, the blasts of acute myeloid leukemia (AML) respond by increasing cellular content of CH, which increases resistance to treatment. (Kornblau et al., 2007) Statins are pharmacologic inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) - the regulatory enzyme of CH synthesis (Nonaka et al., 2009; Sassano et al., 2007). In vitro, they block HMG-CoA reductase and by this they contribute to restore sensitivity to chemotherapy. (Kornblau et al., 2007) HMG-CoA regulates not only the synthesis of CH but also that of the higher isoprenoids, as geranylgeranyl pyrophosphate (Fuchs et al., 2008). By the inhibition of the prenylation processes, in vitro, statins reduce cellular proliferation and stimulate apoptosis of cancerous cells (Nonaka et al., 2009; Sassano et al., 2007). It was found that simvastatin inhibits geranylgeranylation processes of small

Cholesterol and Triglycerides Metabolism Disorder in Malignant Hemopathies 399

In a cell line of acute promyelocytic leukemia (NB4) atorvastatin and fluvastatin showed to be potent stimulators of cell differentiation and apoptosis (Sassano et al., 2007). When to the treatment with idarubicin and high-dose cytarabine of patients with AML pravastatin was added, CR / CRp was observed in 11 of 15 new patients, out of which 8 of 10 had unfavorable cytogenetics, and 9 of 22 patients who received rescue medication that pravastatin did not influence the length of neutropenia, of thrombocytopenia or of the

Statins are active also in acute promyelocytic leukemia cells, where they augment the antileukemic response that depends on all-trans retinoic acid (ATRA). The c-Jun NH2 terminal kinase pathway is required for leukemic cells differentiation induced by statins. Statins also intervene in modulating ATRA-dependent transcription. This was revealed by the selective expression of a large number of genes (400) when atorvastatin was administered together with ATRA. (Sassano et al., 2009) This drug combination could be a

Unlike the subgroup of normal and AML cells CD34 (-), the CD34 (+) is more sensitive to lovastatin. Both populations of cells were strongly inhibited when lovastatin was added to chemotherapy. Leukemic cell samples from different patients with AML had heterogeneous sensitivity to lovastatin. Fifty percent of the patients with unfavorable treatment response had cytogenetic examination with poor prognosis and significantly more blasts in the

It was observed that high expression of CXCR4 correlates with a shorter survival time of patients with AML. In some models of cancer hypoxia it leads to the increase of CXCR4. On the other hand, increased pO(2) causes depletion of CH, which alters lipid rafts and leads to structural changes, which result in increased rejection of CXCR4 microparticles. (Fiegl et al., 2009) Atorvastatin administred in doses of 16 mg/kg body wt showed to be effective in the inhibition of ascites tumor growth and induced apoptosis of a cell line of Daltons'

In vitro, simvastatin induced apoptosis of CLL cells, found in short term culture and contributed to lower BCL-2/BAX report; it was found that its effect of apoptosis induction is tumor-specific and does not affect normal lymphocytes. The association of simvastatin with fludarabine or cladribine synergistically induces DNA damages, and these lead to apoptosis. The proportion of cells found in apoptosis induced by simvastatin +/- chemotherapy was not correlated with the expression of negative prognostic markers of the disease (ZAP-70

The interaction of adhesion molecules, with fundamental role in cellular interaction processes, including those concerning EBV-transformed B cells is blocked by some statins. These drugs also inhibit intracellular activation of NF-kappaB and contribute to the emergence of transformed B-cell apoptosis. In mice with severe combined immune deficiency, simvastatin caused delayed emergence of the lymphomas induced by EBV.

Both the simvastatin and the tipifarnib have cytotoxic effect on AML cell lines and their associated administration has a synergistic effect. This combination administered to CD34(+) AML cells resulted in the increase of the inhibitory effect only on normally responsive AML cells; however, the combination administred to CD34(-) AML cells had

It was observed that statins are able to decrease the expression of BCL-2, an antiapoptotic molecule, favoring the appearance of apoptosis of CD4(+) CD28(null) T cells - a T aggressive

solution for reversing the ATRA-resistance of leukemic cells (Sassano et al., 2007).

Lymphoma Ascites that was transplanted into mice (Ajith et al., 2008).

and CD38) or its stage in the RAI classification. (Podhorecka et al., 2010)

augmented inhibitory effect in all cells. (van der Weide et al., 2009)

toxicity of chemotherapy. (Kornblau et al., 2007)

peripheral blood. (de Jonge-Peeters et al., 2009)

(Cohen et al., 2005)

GTPases Rab5B and Rac1 in certain leukemic cells (for example, adult T-cell leukemia). (Nonaka et al., 2009)

The excessive proliferation inhibition induced by simvastatin results from the induction of apoptosis, cell cycle arrest in phase G2 / M, and accumulation of p21 protein. Simvastatin is able to remove resistance to apoptosis that occurs during treatment with bortezomib, by reducing geranylgeranyl pyrophosphate synthesis and cell survival mechanism dependant on this. (Fuchs et al., 2008) In IgM secreting cell lines and cells from Waldenstrom macroglobulinaemia, simvastatin showed antiproliferative and cytotoxic effects and stimulated the apoptosis. Simvastatin had a synergistic effect with bortezomib, dexamethasone and fludarabine by augmenting their cytotoxicity. (Moreau et al., 2008).

A group of 23 patients with lymphoproliferative diseases, for which statins had not been contraindicated, was treated for 3 days with simvastatin at a dose of 120 mg/day. Serum CH level and that of total lipids decreased significantly (p<0.001 and, respectively, p = 0.016). Serum ALT decreased unsignificantly, while that of AST increased, the growth was close to the statistical significance limit, but was not higher than the upper normal level. Flowcytometric dosage of annexine V showed that simvastatin induced early and late apoptosis increase (p = 0.007, respectively, p = 0.003). By its effect on apoptosis, simvastatin could be an adjuvant treatment for patients with lymphoproliferative disorders. (Mihăilă et al., 2009)

By their CH-lowering effect, statins are promising drugs for the treatment of lymphomas (63 Winiarska et al., 2008). A female patient suffering from NHL with large B-cells whose primary location was the mammary gland had hypercholesterolemia, hypertriglyceridemia and was hypertensive. During the treatment with R-CHOP and radiotherapy that followed (30 Gy), she received lovastatin (20 mg/day) and verapamil. After the first 30 days of treatment, both CH and TG were normalized, and after the whole treatment, the patient has been in complete remission that persists today. (Mihăilă et al, 2008) Lovastatin was administered to the patient not only because she was dyslipidemic, but also because the literature claims that the drug is useful in malignant lymphomas, leukemias and multiple myeloma by its pleiotropic effects. In 1998 the first article about a farnesyltransferase inhibitor (L-744, 832) was published, inhibitor that proved to be effective in mice with mammary carcinomas and lymphomas (Mangues et al., 1998). Lovastatin acts by inhibition of geranylgeranylation, followed by reduction of intracellular signaling mechanisms, which results in reduction of time and dose-dependency of the viability of lymphoma cells in vitro. This is the result of apoptosis stimulation as well as of the decrease of lymphoma cells proliferation, the latter by induction of G1 arrest in cell cycle (van de Donk et al., 2003).

In a rat lymphoma model lovastatin administration during radiotherapy led to cell cycle arrest in different phases, which justified the continuation of the treatment with this drug in the female patient during radiotherapy; the experimental model mentioned, the combination of lovastatin with radiotherapy resulted in a synergistic action (Rozados et al., 2005, as cited in Mihăilă et al., 2008). Lovastatin administration did not preclude the response to polychemoterapy that included rituximab. In fact, although it is only one case, the experimental findings do not always overlap with clinical outcomes. The authors consider that the combination of lovastatin with verapamil favored the response to anticancer treatment and prevented the possible multidrug resistance (Mihăilă et al., 2008). The combination of lovastatin + R-CHOP did not lead to adverse effects. Six years after the end of therapy, the patient is still in complete remission.

GTPases Rab5B and Rac1 in certain leukemic cells (for example, adult T-cell leukemia).

The excessive proliferation inhibition induced by simvastatin results from the induction of apoptosis, cell cycle arrest in phase G2 / M, and accumulation of p21 protein. Simvastatin is able to remove resistance to apoptosis that occurs during treatment with bortezomib, by reducing geranylgeranyl pyrophosphate synthesis and cell survival mechanism dependant on this. (Fuchs et al., 2008) In IgM secreting cell lines and cells from Waldenstrom macroglobulinaemia, simvastatin showed antiproliferative and cytotoxic effects and stimulated the apoptosis. Simvastatin had a synergistic effect with bortezomib, dexamethasone and fludarabine by augmenting their cytotoxicity. (Moreau et al., 2008). A group of 23 patients with lymphoproliferative diseases, for which statins had not been contraindicated, was treated for 3 days with simvastatin at a dose of 120 mg/day. Serum CH level and that of total lipids decreased significantly (p<0.001 and, respectively, p = 0.016). Serum ALT decreased unsignificantly, while that of AST increased, the growth was close to the statistical significance limit, but was not higher than the upper normal level. Flowcytometric dosage of annexine V showed that simvastatin induced early and late apoptosis increase (p = 0.007, respectively, p = 0.003). By its effect on apoptosis, simvastatin could be an adjuvant treatment for patients with lymphoproliferative disorders. (Mihăilă et

By their CH-lowering effect, statins are promising drugs for the treatment of lymphomas (63 Winiarska et al., 2008). A female patient suffering from NHL with large B-cells whose primary location was the mammary gland had hypercholesterolemia, hypertriglyceridemia and was hypertensive. During the treatment with R-CHOP and radiotherapy that followed (30 Gy), she received lovastatin (20 mg/day) and verapamil. After the first 30 days of treatment, both CH and TG were normalized, and after the whole treatment, the patient has been in complete remission that persists today. (Mihăilă et al, 2008) Lovastatin was administered to the patient not only because she was dyslipidemic, but also because the literature claims that the drug is useful in malignant lymphomas, leukemias and multiple myeloma by its pleiotropic effects. In 1998 the first article about a farnesyltransferase inhibitor (L-744, 832) was published, inhibitor that proved to be effective in mice with mammary carcinomas and lymphomas (Mangues et al., 1998). Lovastatin acts by inhibition of geranylgeranylation, followed by reduction of intracellular signaling mechanisms, which results in reduction of time and dose-dependency of the viability of lymphoma cells in vitro. This is the result of apoptosis stimulation as well as of the decrease of lymphoma cells proliferation, the latter by induction of G1 arrest in cell cycle (van de Donk et al., 2003). In a rat lymphoma model lovastatin administration during radiotherapy led to cell cycle arrest in different phases, which justified the continuation of the treatment with this drug in the female patient during radiotherapy; the experimental model mentioned, the combination of lovastatin with radiotherapy resulted in a synergistic action (Rozados et al., 2005, as cited in Mihăilă et al., 2008). Lovastatin administration did not preclude the response to polychemoterapy that included rituximab. In fact, although it is only one case, the experimental findings do not always overlap with clinical outcomes. The authors consider that the combination of lovastatin with verapamil favored the response to anticancer treatment and prevented the possible multidrug resistance (Mihăilă et al., 2008). The combination of lovastatin + R-CHOP did not lead to adverse effects. Six years after the

end of therapy, the patient is still in complete remission.

(Nonaka et al., 2009)

al., 2009)

In a cell line of acute promyelocytic leukemia (NB4) atorvastatin and fluvastatin showed to be potent stimulators of cell differentiation and apoptosis (Sassano et al., 2007). When to the treatment with idarubicin and high-dose cytarabine of patients with AML pravastatin was added, CR / CRp was observed in 11 of 15 new patients, out of which 8 of 10 had unfavorable cytogenetics, and 9 of 22 patients who received rescue medication that pravastatin did not influence the length of neutropenia, of thrombocytopenia or of the toxicity of chemotherapy. (Kornblau et al., 2007)

Statins are active also in acute promyelocytic leukemia cells, where they augment the antileukemic response that depends on all-trans retinoic acid (ATRA). The c-Jun NH2 terminal kinase pathway is required for leukemic cells differentiation induced by statins. Statins also intervene in modulating ATRA-dependent transcription. This was revealed by the selective expression of a large number of genes (400) when atorvastatin was administered together with ATRA. (Sassano et al., 2009) This drug combination could be a solution for reversing the ATRA-resistance of leukemic cells (Sassano et al., 2007).

Unlike the subgroup of normal and AML cells CD34 (-), the CD34 (+) is more sensitive to lovastatin. Both populations of cells were strongly inhibited when lovastatin was added to chemotherapy. Leukemic cell samples from different patients with AML had heterogeneous sensitivity to lovastatin. Fifty percent of the patients with unfavorable treatment response had cytogenetic examination with poor prognosis and significantly more blasts in the peripheral blood. (de Jonge-Peeters et al., 2009)

It was observed that high expression of CXCR4 correlates with a shorter survival time of patients with AML. In some models of cancer hypoxia it leads to the increase of CXCR4. On the other hand, increased pO(2) causes depletion of CH, which alters lipid rafts and leads to structural changes, which result in increased rejection of CXCR4 microparticles. (Fiegl et al., 2009) Atorvastatin administred in doses of 16 mg/kg body wt showed to be effective in the inhibition of ascites tumor growth and induced apoptosis of a cell line of Daltons' Lymphoma Ascites that was transplanted into mice (Ajith et al., 2008).

In vitro, simvastatin induced apoptosis of CLL cells, found in short term culture and contributed to lower BCL-2/BAX report; it was found that its effect of apoptosis induction is tumor-specific and does not affect normal lymphocytes. The association of simvastatin with fludarabine or cladribine synergistically induces DNA damages, and these lead to apoptosis. The proportion of cells found in apoptosis induced by simvastatin +/- chemotherapy was not correlated with the expression of negative prognostic markers of the disease (ZAP-70 and CD38) or its stage in the RAI classification. (Podhorecka et al., 2010)

The interaction of adhesion molecules, with fundamental role in cellular interaction processes, including those concerning EBV-transformed B cells is blocked by some statins. These drugs also inhibit intracellular activation of NF-kappaB and contribute to the emergence of transformed B-cell apoptosis. In mice with severe combined immune deficiency, simvastatin caused delayed emergence of the lymphomas induced by EBV. (Cohen et al., 2005)

Both the simvastatin and the tipifarnib have cytotoxic effect on AML cell lines and their associated administration has a synergistic effect. This combination administered to CD34(+) AML cells resulted in the increase of the inhibitory effect only on normally responsive AML cells; however, the combination administred to CD34(-) AML cells had augmented inhibitory effect in all cells. (van der Weide et al., 2009)

It was observed that statins are able to decrease the expression of BCL-2, an antiapoptotic molecule, favoring the appearance of apoptosis of CD4(+) CD28(null) T cells - a T aggressive

Cholesterol and Triglycerides Metabolism Disorder in Malignant Hemopathies 401

patients expressed P-gp; about 20% of them were positive. Following the administration of lovastatin only 7.33% of them also expressed P-gp (p = 0.016). Compared to the proportion of positive lymphocytes at baseline, the decline was of 63.35%. During the study, CH decreased statistically significantly, with 20.43%. There was no observation of the appearance of possible drug adverse effects. In conclusion, the 6 days therapy with lovastatin was able to reduce significantly the CH and the number of lymphocytes in the membranes where P-gp is expressed, so that this statin could contribute through its pleiotropic effects to reduce multidrug resistance, especially when it is followed by

This drug efflux pump can be inhibited by verapamil, too, the research made in vitro proved that it can reduce multidrug resistance. In such a study conducted in two patients with leukemic lymphoma resistant to treatment, verapamil was able to increase the intracellular

It was found that verapamil was able to overcome the P-gp - mediated resistance to doxorubicin and vincristine in a canine cell line of B cell lymphoma (GL-1) (Uozurmi et al., 2005, as cited in Mihăilă et al., 2008). These experimental findings were not confirmed by parallel administration of chemosensitizer verapamil to chemotherapy (cyclophosphamide, doxorubicin, vincristine, and dexamethasone) in patients with medium and high level NHL found in advanced stages. This drug combination did not increase the therapeutic response and did not extend the survival in these patients as compared to those treated only with the mentioned chemotherapy (without verapamil) (Gaynor et al., 2001, as cited in Mihăilă et al., 2008), but it cannot be excluded that it could be effective in some patients who develop multidrug resistance. But, in metastatic breast carcinoma that has become resistant to anthracyclines verapamil has showed that it is able to increase the survival of patients (Belpomme et al., 2000, as cited in Mihăilă et al., 2008). In the case of the patient described above with hypertension and primary mammary NHL, the evolution has been favorable under the combination of verapamil to chemo- and radiotherapy, that has resulted in a

In another study, 45 patients with proliferative haematological disorders were included in one of the following two groups: A - those who had hypertension and who received verapamil + chemotherapy, and B - those with normal blood pressure, who received only chemotherapy. Group A included 7 patients with chronic lymphoproliferations and 2 with chronic myleoproliferations; under treatment, both systolic and diastolic pressure decreased significantly in all patients in the group. Initially the serum CH level was higher than in the patients in group B (p = 0.004), while other biological tests did not vary significantly between group A and B. No adverse effects were observed during the study. The fact that the initial blood CH was higher in in patients in group A suggests that malignant cells of patients in group B captured more blood CH that contributed to their proliferation. Although the average survival was not significantly different between group A and B, in group A there were more patients with stable disease (77.78% versus 44.44% - p<0.0001) while in group B more patients had progressive disease (30.56 % versus 11.11% - p<0.0001). The deaths due to progressive disease were significantly more numerous in group B. The authors consider that verapamil is useful not only because of its antihypertensive effect, but also due to its pleiotropic effects through which it could influence the evolution of neoplasia by inhibiting P-gp function and the efflux of drugs from lymphoma or leukemia cells.

event-free survival of 6 years (up to the present day) (Mihăilă et al., 2008).

chemotherapy. (Mihăilă et al., 2010)

(Mihăilă et al., 2008)

amount of doxorubicin (Tidefelt et al., 1994).

and long-lasting lymphocyte subpopulation, which can infiltrate the atheromatous plaques, contributing to the their destabilization, which facilitates the instalation of major coronary accidents. (Link et al., 2011)
