F Mentz et al had demonstrated that Theophylline induces apoptosis of B-CLL cells in vitro ₁₅ . They also investigated the synergistic effects of Theophylline and chlorambucil in inducing apoptosis in B-CLL cells and the highest apoptosis values were obtained with 100 µg/mL Theophylline and 10 μM/L chlorambucil. The marker of apoptosis, DNA strand breaks were detected by in situ dUTP labeling, agarose gel electrophoresis and propidium iodide staining in flow cytometry. Methylxanthine derivatives and chlorambucil may thus have potential as a new therapeutic combination in B-CLL.

Hirsh L et al studied that Theophylline (at 15-25 ng/ml) synergizes with gemcitabine or cisplatin to induce programmed cell death in a variety of carcinoma cell lines derived from human ovarian, prostate and lung cancer and in granulosa cell line transformed by SV40 and Ras oncogene. This permits a reduction in the effective doses of cisplatin and gemcitabine by 2-3-fold. The effect of Theophylline in induction of apoptosis involved reduction of intracellular levels of Bcl2.

Yoshida Y et al reported that At concentrations of 0.3 and 1 μM cisplatin, Theophylline at 15 and 50 µg/ml increased the incidence of apoptosis in these cells by 3-5-fold. Bcl-2 protein expression levels were markedly reduced by Theophylline and cisplatin in a dose-dependent manner ₁₆ .

Robert Vassallo et al observed the inhibition of the bcl-2 oncogene expression, and increased the expression of c-myc in Chronic Lymphocytic Leukemic and lymphoma cells by Theophylline. Further, no effect on oncogene expression or rate of apoptosis was demonstrable in the control normal B lymphocytes ₁₇ .

Lentini A et al demonstrated that theophylline and caffeine possess the capacity to inhibit not only cell proliferation, but also the metastatic behavior of melanoma cancer cells ₁₈ .

Przemyslav Janik et al identified that isobutyl methyl xanthine at noncytotoxic concentrations inhibited the tumor colony formation of cloned line Lewis lung carcinoma (LL1) cells in vitro at a concentration of 10 ^-4 M. Administration of IBX as two daily injections beginning 2 days prior to i.v.( IBX dose:5 to 20 mg/kg/injection) LL1 tumor cell inoculation, s.c LL1 inoculation( 10 mg/kg/injection) resulted in a dose-dependent decrease (2- to 10-fold) in formation of lung nodules and 10-fold reduction in the number of lung metastases, smaller lung metastases respectively in C57BL/6J mice ₁₉ .

Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition..Exisulind induced apoptosis by sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction which phosphorylates the accumulated beta –catenin and the phosphorylation of it leads to proteosomal degradation of b-catenin and apoptosis. The cytoplasmic and nuclear beta-catenin accumulations can be prevented by Exisulind in Familial Adenomatous Polyposis (which occurs due to Germ-line mutations in the APC tumor suppressor gene), so it can be considered as a potential therapy in FAP ₂₀ .

Ryosuke Ogawa et al found that glucocorticoid-resistant cell line CEM-CCRF, derived from a child with T-cell ALL. When exposed to methylxanthine derivative, Aminophylline in vitro for 4 days displayed dose-related growth suppression in response to increasing concentrations of it and the fraction of viable cells was measured by the MTT assay. The effective concentration of aminophylline that inhibited viability of treated CEM cells to 50% of control cells (EC50) was 400 μg/mL ₂₁ .

Shulamith H et al proved that Caffeine reduces the DNA synthesis as evidenced by the reduced thymidine uptake as well as the cell viability leading to cell death by its action on the cAMP in the Small cell lung carcinoma cell lines ( NCI-H345, NCI-H128, and SCC-9) ₂₂ .

In a study by Murata et al found that PDE3 plays an important role in the growth of Human submandibular gland intercalated duct cells & PDE3A and 3B mRNAs were detected by reverse transcription-polymerase chain reaction.PDE3 inhibitor such as Cilostamide inhibited the growth of these cells proving that PDE3 as a target for antiproliferative therapy ₂₃ .

DC-TA-46(7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine), PDE-4-specific inhibitor, as reported by Doris Marko et. al., that it arrested the cell cycle in G₀/G₁ phase after 24 hr treatment in the highly malignant Spindle Cell carcinoma cell line(CarB) with highest sensitivity at IC₅₀=0.8±0.3 μM ₂₄ .

In another study by Markus Drees et al observed that DC-TA-46 inhibited growth of MCF-7 mammary carcinoma and B16 melanoma cells in a dose-dependent manner. ₂₅ .

Kohei Murata et al proved that Ro-20-1724, inhibitor for PDE 4 isoenzyme, elevated intracellular cAMP contents three to five times of control & suppressed cell motility (which is assessed by chemotaxis assay) in a dose dependent fashion in a colon cancer cell line (DLD-1). So, PDE 4 can be a novel target of anti-invasion drug.

B Siegmund et al in his study proved that Rolipram (PDE4 inhibitor) induced apoptosis in B-CLL cells at 10 micro M concentrations. The apoptosis-inducing potency of the combination of Rolipram (1 μM) with Fludarabine (1.7 μM) is more than the potency when used alone showing a surprising synergy ₂₆ .

Tiwari S et al found in their observations that Rolipram, synergized with glucocorticoids in inducing B-CLL but not T cell apoptosis augmenting glucocorticoid receptor element (GRE) transactivation mediated by Protein Kinase A activation. This is evidenced by reversal of both glucocorticoid-induced apoptosis and GRE transactivation by cAMP antagonist Rp-8Br-cAMPS, an inhibitor of protein kinase A (PKA) ₂₇ .

Narita et al reported in his findings that HOSM-1 cells(an osteosarcoma cell line established from human mandible) expressed mRNA for PDE4A, 4B and 4C as evidenced by Reverse Transcriptase-PCR analysis and its proliferation was inhibited by Rolipram ₂₈ .

Doo Ho Kim et al observed that when CLL cells were incubated with Rolipram(10 μM/L), forskolin(40 μM/L), or both agents for 72 hrs induced internucleosomal DNA fragmentation characteristic of apoptosis, which is visualised by ethidium bromide ₂₉ .

Huseyin Ustun et al reported that the rats treated with vardenafil and Sildenafil showed significantly increased apoptotic cell death, eNOS(constitutive nitric oxide synthase) and iNOS(indusive NOS) ₃₀ values in ipsilateral testis (P < 0.05).

Paolo Serafi ni et al in their investigations determined immune-mediated antitumor activity of PDE5 inhibitors, using various transplantable mouse tumors such as CT26WT (a colon carcinoma), the more aggressive variant C26GM, TS/A (a mammary adenocarcinoma), and the MCA203 (fibrosarcoma) and PDE5 inhibitors were administered starting on the day of tumor challenge.

To confirm that the antitumor effect of PDE5 inhibitors was immune mediated, the experiments were repeated in immune-compromised BALB/c-Rag-2−/− mice. In these hosts, Sildenafil demonstrated no antitumor efficacy as these mice lacked T and B lymphocytes but have normal or enhanced NK and NKT activity.

Paolo Serafi ni et al found that PDE5 inhibition increases tumor-infiltrating CD8+ T cells, down-regulating arginase 1 and nitric oxide synthase–2expression, there by reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) recruited by growing tumors. By removing these tumor escape mechanisms, PDE5 inhibitors enhance intratumoral T cell infiltration and activation, reducing tumor outgrowth, with increased anti tumor efficacy of adaptive T cell therapy.

Marika Sarfati et al has reported that, Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis and this appeared to be selective for the leukemic B cells because increasing concentrations of either Sildenafil or vardenafil augmented the percentage of annexin V_ cells in B-CLL but not in normal B lymphocytes. Sildenafil and Vardenafil induce B-CLL apoptosis in vitro & it is dependent on caspase activation and is determined using annexin A5 assay and the increased level of active caspase 3 was measured by flow cytometry₁₁ .